Interferon Alpha and Omega Antibody Antagonists

ABSTRACT

The present invention relates to antibodies that broady neutralize interferon-α and interferon-ω, polynucleotides encoding the antibodies or fragments, and methods of making and using the foregoing.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.14/745,939, filed 22 Jun. 2015, currently allowed, which claims thebenefit of U.S. Provisional Application Ser. No. 62/015,765, filed 23Jun. 2014. The entire contents of the aforementioned application areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies that broadly neutralizeinterferon-α and interferon-ω, polynucleotides encoding the antibodiesor fragments, and methods of making and using the foregoing.

BACKGROUND OF THE INVENTION

Type I interferons (IFNs) (IFN-I) are a family of cytokines that signalthrough a ubiquitously expressed heterodimeric receptor IFNAR(heterodimer of IFNAR1 and IFNAR2) resulting in antiviral,antiproliferative and immunomodulatory effects. In humans, type I IFN iscomposed of at least 12 IFN-α protein subtypes and 1 subtype each forIFN-β, IFN-ε, IFN-κ, and IFN-ω. IFN-I release occurs in response to bothmicrobial and sterile ligands. Upon receptor binding, IFN-I initiates asignaling cascade through activation of JAK1 and TYK2 leading to thephosphorylation of several STAT family members including STATs 1-6.STAT1 and STAT2 activation leads to the formation of a complex withIFN-regulatory factor 9 (IRF9) and this complex, also known as theIFN-stimulated gene factor 3 (ISGF3) complex, binds to IFN-stimulatedresponse elements (ISREs) in the nucleus resulting in the transcriptionof many interferon-stimulated genes (ISGs) including IRF7 and CXCL10(IP-10) (Gonzalez-Navajas et al., Nature reviews. Immunology 12, 125(February, 2012). IFN-I also modulates cellular function through otherpathways including the v-crk sarcoma virus CT10 oncogene homolog(avian)-like (CRKL), mitogen-activated protein kinase (MAPK),phosphoinositide 3-kinase (PI3K), and through nuclear factorkappa-light-chain-enhancer of activated B cells (NF-κβ) (Hervas-Stubbset al., Clinical cancer research: an official journal of the AmericanAssociation for Cancer Research 17, 2619 (May 1, 2011)).

Several immune-mediated inflammatory diseases or autoimmune diseases,such as lupus, including Systemic Lupus Erythematosus (SLE) andcutaneous lupus erythematosus (CLE), type I diabetes, psoriasis,Sjögren's disease, systemic sclerosis, rheumatoid arthritis, immunethrombocytopenia (ITP), Aicardi-Goutieres syndrome (AGS), myositis,common variable immune deficiency (CVID) and autoimmune thyroid diseaseare associated at least in a sub-population of patients withoverexpression of IFN-inducible gene transcripts commonly called the IFNsignature present in whole blood and/or tissue, or with elevated IFN-I.

SLE is a chronic autoimmune or immune-mediated inflammatory disease inwhich the production of pathogenic autoantibodies and immune complexesresult in tissue damage across multiple organ systems. The diseasedisplays a broad range of symptoms with heterogeneous clinicalpresentation and may include systemic, cutaneous, renal,musculoskeletal, neurological and hematological manifestations. SLEvaries greatly in severity and is chronic, remitting or relapsing withflares of activity cycling with periods of improvement or remission thatmay last weeks, months, or years. IFN-α is elevated in SLE patients andis believed to promote a loss of tolerance to self. IFN-α has been shownto contribute to sustained dendritic cell activation and thus antigenpresentation, and suppression of Treg function contributing to SLE.IFN-α also induces BLyS expression, a target for the marketed SLEtherapeutic BENLYSTA™. A number of polymorphisms associated withproduction or response to IFN-I have been identified and account forover half of confirmed polymorphisms associated with SLE (Ghodke-Puranik& Niewold, International journal of clinical rheumatology 8,doi:10.2217/ijr.13.58 (2013)). Antibodies neutralizing various IFN-αsubtypes (pan-IFN-α antibodies) are being evaluated in clinical trialsfor SLE (see, for example, Int. Pat. Publ. No. WO02/066649, Int Pat.Publ. No. WO05/059106, Int. Pat. Publ. No. WO06/086586, Int. Pat. Publ.No. WO09/135861).

IFN-ω constitutes approximately 15% of the total IFN-I activity in humanleukocyte IFN preparations produced after viral infection (Adolf,Virology 175, 410 (April, 1990). IFN-ω gene expression has been reportedto be elevated in SLE patients (Han et al., Genes and immunity 4, 177(April, 2003); Yao et al., Hum Genomics Proteomics 2009, (2009)), andthe ability of IFN-ω to induce DC differentiation has been reported(Walker and Tough, European journal of immunology 36, 1827 (July,2006)). The anti-IFN-α antibodies currently in clinical trials(sifalimumab (MEDI-545), rontalizumab and AGS-009) do not neutralizeIFN-ω. Clinical trial data with these antibodies indicate partialreduction of the type I IFN signature in patients after treatment withanti-IFN-α antibodies (Merrill et al., Ann Rheum Dis 70:1905-1913, 2011;Yao et al., Arthritis Rheum 60:1785-1796, 2009), and Phase 2 trial datawith rontalizumab (a pan-anti-IFN-α antibody) indicated improvement insigns and symptoms of SLE, flare rates, and steroid burden at week 24 ina pre-specified biomarker defined group of Interferon Signature Metric(ISM)-Low moderate to severely active lupus subjects. No efficacy wasseen in patients having higher levels of IFN-inducible gene expressionpre-defined as ISM-High (Kalunian et al., 2012 ACR/ARHP Annual Meeting;Abstract #2622, 2012).

In addition to anti-IFN antibodies, anti-IFNAR1 antibodies are beinginvestigated for the treatment of lupus (Wang et al., 2013; ClinicalPharmacology & Therapeutics accepted article preview 14 Feb. 2013; doi:10.1038/clpt.2013.35). IFNAR1 blockage is likely to abolish IFNsignaling induced by all type I IFNs, including IFN-β. IFN-β may play amore critical role in antiviral defense, as specific deletion of thegene encoding IFN-β incurs substantial susceptibility to a host ofviruses when compared to similarly exposed mice having functional IFN-β(Lazear et al., J Virol 85:7186-7194; Deonarain et al., J Virol 74(7):3404-340, 2000; Deonarain et al., Circulation 110: 3540-3543, 2004;Gerlach, et al., J Virol 80: 3438-3444, 2006). Therefore, anti-IFNAR1antibodies may increase the risk of side effects.

Current standard of care for SLE includes corticosteroids, antimalarialdrugs, immunosuppressants or B cell modulators. These therapeutics mayexhibit toxicity and other serious side effects, and may not be suitablefor treatment of all lupus patients. Thus, there is a need foradditional therapeutic treatments for lupus and other immune-mediatedinflammatory or autoimmune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows IFN-ω and IFN-α levels (pg/ml) in plasma from Chinese SLEpatients. Horizontal bars in the figure indicate mean ELISA value ofreplicate samples, vertical bars indicate standard deviation (SD).

FIG. 1B shows IFN-ω and IFN-α levels (pg/ml) in serum from Caucasian SLEpatients. The dark solid circle indicates the highest IFN-α levels andthe dotted line circle indicate the highest IFN-ω plasma levels acrossthe various donors. Horizontal bars in the figure indicate mean ELISAvalue of replicate samples, vertical bars indicate SD.

FIG. 1C shows that patient serum activates downstream interferonsignaling pathways measured using ISRE reporter gene assay. The donorexhibiting the greatest amount of IFN-α protein (dark solid circle) andIFN-ω (dotted line circle) also demonstrated the greatest levels of ISREinduction in the reporter gene assay. The results are readings from asingle well for each serum sample.

FIG. 2 shows inhibition of SLE immune complex-induced IFN withincreasing concentration (0.4-100 μg/ml) of anti-IFN-α antibody alone orat 100 μg/ml in combination with anti-IFN-ω antibody (20 μ/ml). SLEimmune complexes (SLE IC) were prepared from two different donors (SLEDonor 232 or 293). Combined blockage of IFN-α and IFN-ω resulted inenhanced suppression of SLE IC-induced IFN activity, as measured usingthe ISRE assay. HV IC conditioned media=conditioned media from PBMCsstimulated with immune complexes from healthy donors.

FIG. 3 shows induction of IP-10 secretion from PBMCs from 6 healthyindividuals stimulated with IFN-αA or IFN-ω as indicated.

FIG. 4A shows secretion of IFN-γ by CD4⁺ T cells in the presence of DCsdifferentiated in the presence of IFN-ω, IFN-α, IFN-ω and anti-IFN-ωantibody, or IFN-α and anti-IFN-α antibody, or isotype control (iso) asindicated. DCs differentiated in the presence of either IFN-ω or IFN-αinduced activation of CD4⁺ T cells to a same degree, whereas DCsdifferentiated in the presence of anti-IFN-ω or anti-IFN-α neutralizingantibodies did not induce CD4⁺ T cell differentiation. Thedifferentiated DCs were cultured with purified CD4⁺ T cells at DC: CD4⁺T cells ratios of 1:20. Secreted IFN-γ was measured at day 6. Data isrepresentative of 2 studies. Error bars indicate SD of Luminextriplicates. CONC: concentration.

FIG. 4B shows secretion of IL-17 by CD4⁺ T cells in the presence of DCsdifferentiated in the presence of IFN-ω, IFN-α, IFN-ω and anti-IFN-ωantibody, or IFN-α and anti-IFN-α antibody, or isotype control (iso) asindicated. DCs differentiated in the presence of either IFN-ω or IFN-αinduced activation of CD4⁺ T cells to a same degree, whereas DCsdifferentiated in the presence of anti-IFN-ω or anti-IFN-α neutralizingantibodies did not induce CD4⁺ T cell differentiation. Thedifferentiated DCs were cultured with purified CD4⁺ T cells at DC: CD4⁺T cells ratios of 1:20. Secreted IL-17 was measured at day 6. Data isrepresentative of 2 studies. Error bars indicate SD of Luminextriplicates. CONC: concentration.

FIG. 5A shows that IFN-ω) induces T-cell independent B cell activationto the same degree as IFN-α. B cell activation was assessed by CD86surface expression using fluorescently labeled anti-CD86 antibody.T-cell independent B cell activation was induced by CpG (ODN2006) and/oranti-B cell receptor (aBCR) antibodies as indicated in the figure. IFN-ωor IFN-α (IFN-αB2) was used at indicated concentration. Medianfluorescence was measured. B cells were obtained from one donor. Theresults were expressed as mean values of duplicate samples±SD.

FIG. 5B shows that IFN-ω induces IL-6 secretion from B cells activatedin non-T cell dependent fashion to the same degree as IFN-α. T-cellindependent B cell activation was induced by CpG (ODN-2006) and/oranti-BCR antibodies (aBCR) as indicated in the figure. IFN-ω or IFN-α(IFN-α2B) was used at indicated concentration. IL-6 concentration isindicated as pg/ml. B cells were obtained from one donor. The resultswere expressed as mean values of duplicate samples±SD.

FIG. 6 shows that IFN-ω induces BLyS secretion from human PBMCs to thesame degree as IFN-α (IFN-αB2). The concentration of IFN-ω or IFN-α usedto stimulate PBMCs is indicated in the X-axis. BLyS concentration isshown as pg/ml. Results are expressed as mean values of duplicatesamples±SD.

FIG. 7A shows the overall molecular structure of the IFN-ω/Fab IFWM371complex (only the Fv for the antibody is shown). The boxed area ismagnified in FIG. 7B. IFN-ω AB loop (AB), E helix (E) and D helix (D) ofIFN-ω are indicated. Small circles represent water molecules. VL and VHor IFWM371 are indicated.

FIG. 7B shows a magnification of the boxed area of FIG. 7A,demonstrating hydrogen bonding network mediated through water molecules(water complex (WC) 1, 2, and 3) at the IFN-ω/Fab IFWM371interface.

FIG. 8A shows the epitope in the IFN-ω/Fab IFWM371 complex. IFN-ωresidue numbering according to SEQ ID NO: 1.

FIG. 8B shows the paratope in the IFN-ω/Fab IFWM371 complex. ResiduesY32, Y92, T94 and L96 are residues in the VL, and residues W33, I50,D57, T58, R59, H99, P100, G101, L102, N103, W104, A105 and D107 areresidues in the VH in contact with IFN-ω. VL: SEQ ID NO: 29; VH: SEQ IDNO: 28. T94 and A105 are not shown in the figure.

FIG. 8C shows a 2-dimensional interaction map between IFN-ω and FabIFWM371. Boxed residues are VL paratope residues, and circled residuesare VH paratope residues. Residues highlighted in gray are IFN-ω)epitope residues. Numbering of VL, VH and IFN-ω residues is according toSEQ ID NOs: 29, 28 and 1, respectively. Van der Walls (VDW) andhydrophobic interactions are shown in solid lines, electrostatic and Hbonds in dashed lines, arrows indicate backbone interactions with thearrows pointing to the backbone atoms. Most interactions are formed bythe three IFN-ω epitope residues F27, L30 and R33.

FIG. 9 shows an alignment of IFN-ω with various IFN-α subtypes. Arrowsindicate epitope residues IFWM371 binds to. F27, L30 and R33 areconserved across Type I IFNs, except in IFN-αD to which IFWM371 does notbind to. Residue numbering is according to human IFN-ω SEQ ID NO: 1(IFNω-01 in the Figure). IFNα-01/D/1: SEQ ID NO: 18; IFNα-02/A: SEQ IDNO: 5; IFNα-04/a/b: SEQ ID NO: 15; IFNα-07/J: SEQ ID NO: 13; IFNα-10/C:SEQ ID NO: 7; IFNα-17/I: SEQ ID NO: 12; IFNα-21/F: SEQ ID NO: 9;IFNα-14/H: SEQ ID NO: 11; IFNα-16/WA: SEQ ID NO: 16; IFNα-08/B2: SEQ IDNO: 6; IFNα-05/G: SEQ ID NO: 10; IFNα-06/K: SEQ ID NO: 14.

FIG. 10 shows the IC₅₀ values for select antibodies to various Type IIFNs in an ISRE assay.

FIG. 11A shows neutralization of leukocyte IFN-induced IP10 release inhuman whole blood with anti-IFN-α/ω antibodies. Leukocyte IFN (Lk) wasused to induce IP-10 secretion in healthy donor whole blood from 2subjects. Whole blood was incubated with leukocyte interferon (LK) withor without anti-IFN-α/ω antibodies IFWM3522 or IFWM3525 at variousconcentrations (10 μg/ml-10 pg/ml) as indicated in the Figure. Barrepresents mean and error bars SD from duplicate wells. Data isrepresentative result of 2 independent experiments using whole bloodfrom 2 different human donors.

FIG. 11B shows neutralization of leukocyte IFN-induced IP-10 release inhuman whole blood with anti-IFN-α/ω antibodies. Leukocyte IFN (Lk) wasused to induce IP-10 secretion in healthy donor whole blood from 2subjects. Whole blood was incubated with leukocyte interferon (LK) withor without anti-IFN-α/ω antibody IFWM3399 or isotype control at variousconcentrations (10 μg/ml-10 pg/ml) as indicated in the Figure. Barrepresents mean and error bars SD from duplicate wells. Data isrepresentative result of 2 independent experiments using whole bloodfrom 2 different human donors.

FIG. 12A shows neutralization of SLE immune complex-induced IP-10release in human whole blood with anti-IFN-α/ω antibodies. Whole bloodwas incubated with SLE immune complex-induced interferon preparationswith or without anti-IFN-α/ω antibodies IFWM3522 or IFWM3525 at variousconcentrations (10 μg/ml-10 pg/ml) as indicated in the Figure, and IP-10was analyzed from plasma using an ELISA kit. Bar represents mean anderror bars SD from duplicate wells. Data is representative result of 4independent experiments using whole blood from 2 different human donors.

FIG. 12B shows neutralization of SLE immune complex-induced IP-10release in human whole blood with anti-IFN-α/ω antibodies. Whole bloodwas incubated with SLE immune complex-induced interferon preparationswith or without anti-IFN-α/ω antibody IFWM3399 or isotype control atvarious concentrations (10 μg/ml-10 pg/ml) as indicated in the Figure,and IP-10 was analyzed from plasma using an ELISA kit. Bar representsmean and error bars SD from duplicate wells. Data is representativeresult of 4 independent experiments using whole blood from 2 differenthuman donors.

FIG. 13A shows normalization of MX1 gene expression in SLE patient bloodafter in vitro exposure of the blood to IFN-α/ω antibody IFWM2423 orisotype control for 24 hours at various concentrations (μg/ml) asindicated in the Figure. Bar represents mean and error bars SD fromtriplicate wells. MX1 gene expression was normalized to β-actin.

FIG. 13B shows normalization of MX1 gene expression in SLE patient bloodafter in vitro exposure of the blood to IFN-α/ω antibody IFWM3522 andIFWM2525 or isotype control for 24 hours at various concentrations(μg/ml) as indicated in the Figure. Bar represents mean and error barsSD from triplicate wells. MX1 gene expression was normalized to β-actin.

FIG. 14A shows the hydrogen (H) bond interactions between epitoperesidue R33 with VH of M371 as well as water molecules at theantibody/antigen interface in the IFN-ω/M341 structure.

FIG. 14B shows modified H bond interactions between epitope residue R33with VH of M3421 as well as water molecules in IFN-ω/M3421 structure.

FIG. 14C shows the sequence (L961 mutation) and structural changes uponmaturation of M371. In the M371 structure, F108 of VH is best describedas having two alternative conformations. In the M3421 structure, theyare converted into one conformation, suggesting tighter packing betweenVH and VL. In addition, there is a side chain rotamer flip of the W47 ofVH.

FIG. 14D shows sequence and structural changes upon M371 maturation. TheVL Y32 was mutated into a more hydrophobic F (Y32F) and removing the twoH bonds between Y32 in M371 and IFN-ω. VL A50 was mutated into F (A50F).This residue does not directly contact the antigen but stacks againstW104 of VH that contacts the antigen. Two other changes (S31G and S30D)are not involved in antigen binding or directly impacting bindingresidues like A50F. These residue changes are likely to influence localhydrophobicity and optimize solvent interaction.

FIG. 15 shows s a 2-dimensional interaction map between IFN-ω and FabIFWM3421. Boxed residues are VL paratope residues, and circled residuesare VH paratope residues. Residues highlighted in gray are IFN-ω epitoperesidues. Numbering of VL, VH and IFN-ω residues is according to SEQ IDNOs: 28, 71 and 1, respectively. Van der Waals (VDW) and hydrophobicinteractions are shown in solid lines, electrostatic and H bonds indashed lines, arrows indicate backbone interactions with the arrowspointing to the backbone atoms. Most interactions are formed by thethree IFN-ω epitope residues F27, L30 and R33.

FIG. 16 shows s a 2-dimensional interaction map between IFN-ω and Fab ofIFWM3525. Boxed residues are VL paratope residues, and circled residuesare VH paratope residues. Residues highlighted in gray are IFN-ω epitoperesidues. Numbering of VL, VH and IFN-ω residues is according to SEQ IDNOs: 28, 71 and 1, respectively. Van der Waals (VDW) and hydrophobicinteractions are shown in solid lines, electrostatic and H bonds indashed lines, arrows indicate backbone interactions with the arrowspointing to the backbone atoms. Most interactions are formed by thethree IFN-ω epitope residues F27, L30 and R33.

SUMMARY OF THE INVENTION

One embodiment of the invention is an isolated monoclonal antibody thatbinds to and neutralizes a biological activity of a human interferonomega (IFN-ω) and at least three, four, five, six, seven, eight, nine,ten or eleven human interferon alpha (IFN-α) subtypes.

Another embodiment of the invention is an isolated monoclonal antibodythat binds to and neutralizes a biological activity of a humaninterferon omega (IFN-ω) and at least three, four, five, six, seven,eight, nine, ten or eleven human interferon alpha (IFN-α) subtypes,wherein the antibody neutralizes the biological activity of the humanIFN-ω with an IC₅₀ of at least about 1×10⁻⁹M or less, about 1×10⁻¹¹ M orless, about 5×10⁻¹¹M or less, or about 1×10⁻¹¹M or less.

In other embodiments, the antibody of the invention neutralizes theactivity of at least three, four, five, six, seven, eight, nine, ten oreleven human IFN-α subtypes with an IC₅₀ value of at least about 2×10⁻¹⁰M or less, about 1.5×10⁻¹⁰ M or less, or about 1×10⁻¹° M or less.

In other embodiments, the antibody comprises heavy chain complementaritydetermining region (HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) amino acidsequences of SEQ ID NOs: 109, 114 and 121, respectfully, and light chaincomplementarity determining region (LCDR) 1 (LCDR1), 2 (LCDR2) and 3(LCDR3) amino acid sequences of SEQ ID NOs: 118, 119 and 120.

In other embodiments, the antibody comprises the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2 and the LCDR3 amino acid sequences of SEQ IDNOs: 109, 114, 121, 159, 119 and 160, respectively.

In other embodiments, the antibody neutralizes at least ten human IFN-αsubtypes selected from the group consisting of IFN-αA, IFN-αB2, IFN-αC,IFN-αF, IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK, IFN-αWA and IFN-α4a.

In other embodiments, the antibody binds human IFN-ω of SEQ ID NO: 1 atleast at amino acid residues F27, L30 and R33.

In other embodiments, the antibody comprises the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2 and the LCDR3 amino acid sequences of SEQ IDNOs: 109, 114, 121, 161, 119 and 162, respectively.

In other embodiments, the antibody neutralizes at least the human IFN-αsubtypes IFN-αA, IFN-αB2, IFN-αC, IFN-αF, IFN-αG, IFN-αH2, IFN-αJ1 andIFN-α4a.

In other embodiments, the antibody comprises a heavy chain variableregion (VH) amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 28 and a light chainvariable region (VL) amino acid sequences at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 150.

In other embodiments, the antibody comprises certain HCDR and LCDRsequences as described herein.

In other embodiments, the antibody comprises certain VH and VL sequencesas described herein.

Another embodiment of the invention is a pharmaceutical compositioncomprising the antibody of the invention and a pharmaceutically acceptedcarrier.

Another embodiment of the invention is a polynucleotide encoding theantibody VH and/or the VL of the invention.

Another embodiment of the invention is a vector comprising thepolynucleotide of the invention.

Another embodiment of the invention is a host cell comprising the vectorof the invention.

Another embodiment of the invention is a method of producing theantibody of the invention, comprising culturing the host cell of theinvention in conditions that the antibody is expressed, and recoveringthe antibody produced by the host cell.

Another embodiment of the invention is a method of treating animmune-mediated inflammatory disease or an autoimmune disease,comprising administering a therapeutically effective amount of anisolated antibody of the invention to a patient in need thereof for atime sufficient to treat or prevent the disease.

In some embodiments, the immune-mediated inflammatory disease or theautoimmune disease is lupus, psoriasis, immune thrombocytopenia (ITP),Aicardi-Goutieres syndrome (AGS), systemic sclerosis, Sjögren'ssyndrome, myositis, common variable immune deficiency (CVID), autoimmunethyroid disease, type I diabetes, rheumatoid arthritis, transplantrejection or graft versus host disease (GVHD).

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention pertains.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentinvention, exemplary materials and methods are described herein. Indescribing and claiming the present invention, the following terminologywill be used.

The term “specific binding” or “specifically binds” or “binds” as usedherein refers to antibody binding to an antigen or an epitope within theantigen with greater affinity than for other antigens. Typically, theantibody binds to the antigen or the epitope within the antigen with adissociation constant (K_(D)) of 1×10⁻⁸ M or less, for example 1×10⁻⁹ Mor less, 1×10⁻¹⁰ M or less, 1×10⁻¹¹ M or less, or 1×10⁻¹² M or less,typically with a K_(D) that is at least ten fold less than its K_(D) forbinding to a non-specific antigen (e.g., BSA, casein). The dissociationconstant can be measured using standard procedures. Antibodies thatspecifically bind to the antigen or the epitope within the antigen may,however, have cross-reactivity to other related antigens, for example tothe same antigen from other species (homologs), such as human or monkey,for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes(chimpanzee, chimp). Antibodies that specifically bind to the antigen orthe epitope within the antigen can further bind an epitope that isshared between two or more distinct antigens such as at least oneinterferon alpha (IFN-α) subtype and interferon omega (IFN-ω); i.e.antibodies cross-react with IFN-α subtypes and IFN-ω.

The term “neutralizing” or “neutralizes” or “neutralizing antibody” or“antibody antagonist” as used herein refers to an antibody or antibodyfragment that partially or completely inhibits biological activity ofrecombinant human interferon omega (IFN-ω) and/or at least onerecombinant human interferon alpha (IFN-α) subtype. Neutralizingantibodies may be identified using assays for IFN-α and/or IFN-ω)biological activity as described herein. IFN-α and/or IFN-ω)neutralizing antibody may inhibit measured IFN-α and/or IFN-ω biologicalactivity by 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100%.

The term “interferon-α” (IFN-α) as used herein refers to all nativesubtypes of human alpha interferons. Native IFN-α consists of at least12 closely related protein subtypes encoded by distinct genes with ahigh degree of structural homology (Weissmann and Weber, Prog Nucl AcidRes Mol Biol., 33: 251, 1986; Roberts et al., J Interferon Cytokine Res.18: 805-816, 1998). Nomenclature for human interferons is found at:http://www_genenames_org/genefamilies/_IFN. Table 4 shows the sequencesof the IFN-α subtypes used herein, in addition to other Type I IFNs.

The term IFN-ω) as used herein refers to human IFN-ω) having the aminoacid sequence shown in SEQ ID NO: 1 and UniProt accession number P05000.Human IFN-ω) also includes the variant of SEQ ID NO: 2 having athreonine to glutamic acid substitution at position 80 (T80).

The term “type I interferon” or “IFN-I” refers to all native subtypes ofhuman interferon-α and one subtype of interferon-0, interferon-s,interferon-w and interferon-K which bind to a common interferon receptorIFNAR.

As used herein the term “IFNAR” refers to the well-known interferonreceptor which is a heterodimer or IFNAR1 and IFNAR2. IFNAR1 and IFNAR2protein sequences are shown in SEQ ID NOs: 26 and 27, respectively.IFNAR1 mature extracellular domain spans residues 28-436 of SEQ ID NO:26 and IFNAR2 mature extracellular domain spans residues 27-243 of SEQID NO: 27.

The term “antibodies” as used herein is meant in a broad sense andincludes immunoglobulin molecules including polyclonal antibodies,monoclonal antibodies including murine, human, humanized and chimericmonoclonal antibodies, antibody fragments, bispecific or multispecificantibodies formed from at least two intact antibodies or antibodyfragments, dimeric, tetrameric or multimeric antibodies, single chainantibodies, domain antibodies and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity.

Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE,IgG and IgM, depending on the heavy chain constant domain amino acidsequence. IgA and IgG are further sub-classified as the isotypes IgA₁,IgA₂, IgG₁, IgG₂, IgG₃ and IgG₄. Antibody light chains of any vertebratespecies can be assigned to one of two clearly distinct types, namelykappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

The term “antibody fragments” refers to a portion of an immunoglobulinmolecule that retains the heavy chain and/or the light chain antigenbinding site, such as heavy chain complementarity determining regions(HCDR) 1, 2 and 3, light chain complementarity determining regions(LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chainvariable region (VL). Antibody fragments include well known Fab,F(ab′)2, Fd and Fv fragments as well as domain antibodies (dAb)consisting one VH domain. VH and VL domains can be linked together via asynthetic linker to form various types of single chain antibody designswhere the VH/VL domains pair intramolecularly, or intermolecularly inthose cases when the VH and VL domains are expressed by separate singlechain antibody constructs, to form a monovalent antigen binding site,such as single chain Fv (scFv) or diabody; described for example in Int.Pat. Publ. No. WO1998/44001, Int. Pat. Publ. No. WO1988/01649; Int. Pat.Publ. No. WO1994/13804; Int. Pat. Publ. No. WO1992/01047.

An antibody variable region consists of a “framework” region interruptedby three “antigen binding sites”. The antigen binding sites are definedusing various terms: (i) Complementarity Determining Regions (CDRs),three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1,LCDR2, LCDR3), are based on sequence variability (Wu and Kabat, J ExpMed 132:211-50, 1970; Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991). (ii) “Hypervariableregions”, “HVR”, or “HV”, three in the VH (H1, H2, H3) and three in theVL (L1, L2, L3), refer to the regions of an antibody variable domainswhich are hypervariable in structure as defined by Chothia and Lesk(Chothia and Lesk, Mol Biol 196:901-17, 1987). Other terms include“IMGT-CDRs” (Lefranc et al., Dev Comparat Immunol 27:55-77, 2003) and“Specificity Determining Residue Usage” (SDRU) (Almagro, Mol Recognit17:132-43, 2004). The International ImMunoGeneTics (IMGT) database(http://www_imgt_org) provides a standardized numbering and definitionof antigen-binding sites. The correspondence between CDRs, HVs and IMGTdelineations is described in Lefranc et al., Dev Comparat Immunol27:55-77, 2003.

“Monoclonal antibody” as used herein refers to a homogenous antibodypopulation with singular molecular composition. Monoclonal antibody maybe nonspecific or multispecific.

“Chothia residues” as used herein are the antibody VL and VH residuesnumbered according to Al-Lazikani (Al-Lazikani et al., J Mol Biol273:927-48, 1997).

“Framework” or “framework sequences” are the remaining sequences of avariable region other than those defined to be antigen binding site.Because the antigen binding site can be defined by various terms asdescribed above, the exact amino acid sequence of a framework depends onhow the antigen-binding site was defined.

“Humanized antibodies” refers to antibodies in which the antigen bindingsites are derived from non-human species and the variable regionframeworks are derived from human immunoglobulin sequences. Humanizedantibodies may include substitutions in the framework regions so thatthe framework may not be an exact copy of expressed human immunoglobulinor germline gene sequences.

“Human-adapted” antibodies or “human framework adapted (HFA)” antibodiesrefers to humanized antibodies adapted according to methods described inU.S. Pat. Publ. No. US2009/0118127. Human-adapted antibodies arehumanized by selecting the acceptor human frameworks based on themaximum CDR and FR similarities, length compatibilities and sequencesimilarities of CDR1 and CDR2 loops and a portion of light chain CDR3loops.

“Human antibody” refers to an antibody having heavy and light chainvariable regions in which both the framework and the antigen bindingsite regions are derived from sequences of human origin. If the antibodycontains a constant region, the constant region also is derived fromsequences of human origin.

Human antibody comprises heavy or light chain variable regions that are“derived from” sequences of human origin if the variable regions of theantibody are obtained from a system that uses human germlineimmunoglobulin or rearranged immunoglobulin genes. Such exemplarysystems are human immunoglobulin gene libraries displayed on phage, andtransgenic non-human animals such as mice carrying human immunoglobulinloci as described herein. “Human antibody” may contain amino aciddifferences when compared to the human germline or rearrangedimmunoglobulin sequences due to for example naturally occurring somaticmutations or intentional introduction of substitutions. Typically,“human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%% identical in amino acid sequence to an amino acid sequence encoded bya human germline or rearranged immunoglobulin gene. In some cases,“human antibody” may contain consensus framework sequences derived fromhuman framework sequence analyses, for example as described in Knappiket al (2000) J Mol. Biol. 296:57-86), or synthetic HCDR3 incorporatedinto human immunoglobulin gene libraries displayed on phage, for exampleas described in Shi et al (2010) J. Mol. Biol. 397:385-96, 2010 and Int.Pat. Publ. No. WO2009/085462.

Isolated humanized antibodies are synthetic. Human antibodies, whilederived from human immunoglobulin sequences, may be generated usingsystems such as phage display incorporating synthetic CDRs and/orsynthetic frameworks, or can be subjected to in vitro mutagenesis toimprove antibody properties, resulting in antibodies that do notnaturally exist within the human antibody germline repertoire in vivo.

Human antibodies may include substitutions in the framework or in theantigen binding site so that they may not be exact copies of expressedhuman immunoglobulin or germline gene sequences. However, antibodies inwhich antigen binding sites are derived from a non-human species are notincluded in the definition of “human antibody”.

The term “recombinant” as used herein, includes antibodies and otherproteins, such as various IFN-α subtypes or IFN-ω that are prepared,expressed, created or isolated by recombinant means.

The term “epitope” as used herein means a portion of an antigen to whichan antibody specifically binds. Epitopes usually consist of chemicallyactive (such as polar, non-polar or hydrophobic) surface groupings ofmoieties such as amino acids or polysaccharide side chains and can havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. An epitope can be composed ofcontiguous and/or discontiguous amino acids that form a conformationalspatial unit. For a discontiguous epitope, amino acids from differingportions of the linear sequence of the antigen come in close proximityin 3-dimensional space through the folding of the protein molecule.

“Bispecific” as used herein refers to an antibody that binds twodistinct antigens or two distinct epitopes within an antigen. Thebispecific antibody may have cross-reactivity to other related antigensor can bind an epitope that is shared between two or more distinctantigens such as at least one IFN-α subtype and IFN-ω.

The term “in combination with” as used herein means that the drugs ortherapeutics can be administered to an animal species such as humantogether in a mixture, concurrently as single agents or sequentially assingle agents in any order.

The terms “IFN-α biological activity” and “IFN-ω biological activity” asused herein refer to any activity occurring as a result of IFN-α andIFN-ω, respectively, binding to its receptor IFNAR. One IFN-α and IFN-ωbiological activity is the ability of IFN-α and IFN-ω to induce secretedembryonic alkaline phosphatase (SEAP) expression under the interferoninducible promoter such as ISG54 in HEK293 cells stably expressingsignal transducer and activator of transcription 2 (STAT2), interferonregulatory factor 9 (IRF9) and SEAP using standard methods. AnotherIFN-α and IFN-ω biological activity is the induction of chemokine IP-10(CXCL10) production from peripheral blood mononuclear cells (PBMCs) orwhole blood as described herein.

The term “vector” means a polynucleotide capable of being duplicatedwithin a biological system or that can be moved between such systems.Vector polynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers, that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

The term “expression vector” means a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

The term “polynucleotide” means a molecule comprising a chain ofnucleotides covalently linked by a sugar-phosphate backbone or otherequivalent covalent chemistry. Double and single-stranded DNAs and RNAsare typical examples of polynucleotides.

The term “polypeptide” or “protein” means a molecule that comprises atleast two amino acid residues linked by a peptide bond to form apolypeptide. Small polypeptides of less than 50 amino acids may bereferred to as “peptides”.

Conventional one and three-letter amino acid codes are used herein asshown in Table 1.

TABLE 1 Three- One- Amino letter letter acid code code Alanine ala AArginine arg R Asparagine asn N Aspartate asp D Cysteine cys C Glutamateglu E Glutamine gln Q Glycine gly G Histidine his H Isoleucine ile ILeucine leu L Lysine lys K Methionine met M Phenylalanine phe F Prolinepro P Serine ser S Threonine thr T Tryptophan trp W Tyrosine tyr YValine val V

Compositions of Matter

The present invention provides monoclonal antibodies that bind to andneutralize activity of human interferon omega (IFN-ω) and multiple humaninterferon alpha (IFN-α) subtypes (anti-IFN-α/ω antibodies). Theinvention is based on, at least part, in the appreciation of the role ofINF-ω in lupus pathogenesis with similar immunomodulatory effects thanthose of IFN-α alone. IFN-ω was found to be present and active in serumof lupus patients, and IFN-ω was found to induce similar cytokinerelease and gene expression profiles, dendritic cell differentiation,and T-cell independent B cell activation when compared to IFN-α;providing the basis for the rationale for neutralizing both IFN-α andIFN-ω to maximize therapeutic effect. The invention is also based, atleast in part, on the identification of a minimal neutralizing epitopeshared by IFN-ω and multiple IFN-α subtypes to which the IFN-α/ωantibodies of the invention bind. The IFN-α/ω antibodies of theinvention may neutralize IFN-ω and multiple IFN-α subtypes with highefficacy, and thus they may be more potent in neutralizing SLE-relevantpreparations of type I IFN and IFN signatures than antibodiesneutralizing multiple IFN-α subtypes but not IFN-ω. Therefore, theantibodies of the invention may be more efficacious in treatingimmune-mediated inflammatory diseases or autoimmune diseases includinglupus. As the IFN-α/o antibodies of the invention do not neutralizeIFN-β, they may have more favorable safety and PK profiles when comparedto the anti-IFNAR therapies, which are expected to block all type IIFNs.

One embodiment of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow is an isolated monoclonal antibody that binds to and neutralizes abiological activity of a human interferon omega (IFN-ω) and at leastthree, four, five, six, seven, eight, nine, ten or eleven humaninterferon alpha (IFN-α) subtypes.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes the activity of the human IFN-ω with an IC₅₀of at least about 1×10⁻⁹M or less, about 1×10⁻¹⁰ M or less, about5×10⁻¹¹M or less, or about 1×10⁻¹¹M or less, when the activity of thehuman IFN-ω is the human IFN-ω-induced expression of secreted embryonicalkaline phosphatase (SEAP) under interferon inducible ISG54 promoter inHEK293 cells stably expressing signal transducer and activator oftranscription 2 (STAT2), interferon regulatory factor 9 (IRF9) and SEAP(“ISRE assay” as described herein).

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and at least three, four, five, six,seven, eight, nine, ten or eleven human interferon alpha (IFN-α)subtypes selected from the group consisting of IFN-αA, IFN-αB2, IFN-αC,IFN-αF, IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK, IFN-αWA and IFN-α4a.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αH2 and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αG, IFN-αH2 and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αF, IFN-αG, IFN-αH2 and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αF, IFN-αG, IFN-αH2 andIFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αF, IFN-αG, IFN-αH2,IFN-αJ1 and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αG, IFN-αH2 andIFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αF, IFN-αG,IFN-αH2 and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αC, IFN-αG,IFN-αH2 and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αC, IFN-αF,IFN-αG and IFN-α4a.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αF, IFN-αG,IFN-αH2, IFN-αI and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αF, IFN-αG,IFN-αH2, IFN-αJ1 and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αC, IFN-αF,IFN-αG, IFN-αH2, IFN-αJ1 and IFN-αK.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αC, IFN-αF,IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK and IFN-α4a.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αC, IFN-αF,IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αWA and IFN-α4a.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αC, IFN-αF,IFN-αG, IFN-αH2, IFN-αK, IFN-αWA and IFN-α4a.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes IFN-ω and IFN-αA, IFN-αB, IFN-αC, IFN-αF,IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK, IFN-αWA and IFN-α4a.

Antibodies of the invention described herein, and in some embodiments ofeach and every one of the numbered embodiments listed below, may bindand neutralize at least three, four, five, six, seven, eight, nine, tenor eleven IFN-α subtypes in addition to neutralizing IFN-ω. The IFN-αsubtypes and IFN-ω may be produced by recombinant expression usingstandard methods. Exemplary signal sequences that can be used fordirecting secretion are shown in SEQ ID NOs: 21-25.

The antibodies of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, may be tested for their ability to neutralize IFN-α and IFN-ω ina reporter gene assay using cell lines expressing reporter genes underan interferon responsive promoter, and stimulating cells with variousIFN-α subtypes and/or IFN-ω. For example, HEK-Blue™ IFN-α/β cells(InvivoGen, San Diego, Calif.) engineered to express a fully active typeI IFN signaling pathway (stably expressing STAT2 and IRF9) andtransfected with a SEAP reporter gene under the control of the IFNα/βinducible ISG54 promoter can be used as described herein. Signal fromthe alkaline phosphatase may be detected an IC₅₀ may be calculated forthe inhibition using well known methods.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodiesof the invention neutralize the biological activity of the human IFN-ωwith an IC₅₀ value of about 1×10⁻⁹ M or less, about 1×10⁻¹⁰ M or less,about 5×10⁻¹¹M or less, or about 1×10⁻¹¹M or less, when the biologicalactivity of the human IFN-ω is inhibition of secreted embryonic alkalinephosphatase (SEAP) expression under the interferon inducible ISG54promoter in HEK293 cells stably expressing signal transducer andactivator of transcription 2 (STAT2), interferon regulatory factor 9(IRF9) and SEAP, using the assay “ISRE reporter gene assay” as describedherein in Example 1.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodiesof the invention neutralize the biological activity of the human IFN-ωwith an IC₅₀ value of at least about 1×10⁻¹⁰ M or less, when the IC₅₀ ismeasured in the “ISRE reporter gene assay” described herein.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodiesof the invention neutralize the biological activity of the human IFN-ωwith an IC₅₀ value between about 1×10⁻¹⁰ M to about 6×10⁻¹²M, when theIC₅₀ is measured in the “ISRE reporter gene assay” described herein.Skilled in the art will appreciate that the assay deviation for the ISREreporter gene assay may typically be approximately within pIC₅₀ of about0.28 (log (M)). Therefore the term “about” reflects the typical standarddeviation in the assay. For example, the typical SD for an IC₅₀ of1×10⁻⁹ M is between about 0.53×10⁻⁹ to 1.9×10⁻⁹.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodiesof the invention neutralize the biological activity at least three,four, five, six, seven, eight, nine, ten or eleven human IFN-α subtypeswith an IC₅₀ value of at least about 2×10⁻¹⁰ M or less, about 1.5×10⁻¹⁰M or less, or about 1×10⁻¹⁰ M or less.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes the activity of the human IFN-ω with an IC₅₀value of at least about 1×10⁻¹⁰ M or less, and at least 6 human IFN-αsubtypes with an IC₅₀ value of about 2×10⁻¹⁰ M or less, about 1.5×10⁻¹⁰M or less, or about 1×10⁻¹⁰ M or less, when the IC₅₀ value is measuredusing the “ISRE reporter gene assay” described herein.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes the activity of the human IFN-ω with an IC₅₀value of at least about 1×10⁻¹⁰ M or less, and at least 10 human IFN-αsubtypes with an IC₅₀ value of about 2×10⁻¹⁰ M or less, about 1.5×10⁻¹⁰M or less, or about 1×10⁻¹⁰ M or less, when the IC₅₀ value is measuredusing the “ISRE reporter gene assay” described herein.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes the activity of the human IFN-ω with an IC₅₀value of at least about 1×10⁻¹⁰ M or less, and at least 6 human IFN-αsubtypes with an IC₅₀ value of about 1×10⁻¹⁰ M or less, when the IC₅₀value is measured using the “ISRE reporter gene assay” described herein.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes the activity of the human IFN-ω with an IC₅₀value of at least about 1×10⁻¹⁰ M or less, and at least 10 human IFN-αsubtypes with an IC₅₀ value of about 1×10⁻¹⁰ M or less, when the IC₅₀value is measured using the “ISRE reporter gene assay” described herein.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodiesof the invention inhibit leukocyte interferon-induced IP-10 release inwhole blood induced by 250 U/ml of interferon by about 50% or more inthe presence of 10 μg/ml antibody than in the absence of the antibody.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodiesof the invention inhibit systemic lupus erythematosus (SLE) immunecomplex-induced IP-10 release in whole blood by about 50% or more in thepresence of 10 μg/ml antibody than in the absence of the antibody.

Antibodies of the invention described herein, and in some embodiments ofeach and every one of the numbered embodiments listed below, can betested for their neutralizing ability by assessing their ability toinhibit IFN-induced cytokine release, such as IP-10 release fromIFN-induced peripheral blood mononuclear cells (PBMCs) or whole blood.For example, PBMCs are isolated from heparinized whole blood fromhealthy volunteers using standard protocols, treated with a preformedcomplex of IFN and antibody to be tested, and IP-10 release is measuredusing standard methods such as Milliplex cytokine/chemokine kit(Millipore, Premixed 39 plex). Antibodies of the invention may inhibitIP-10 release by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% when compared to IFN-induced IP-10release in the absence of the antibody.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodiesof the invention bind human IFN-ω) with a dissociation constant (K_(D))of about 1×10⁻¹⁰ M or less, about 5×10⁻¹¹ M or less, about 1×10⁻¹¹ M orless or about 5×10⁻¹² M or less.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention binds IFN-ω) and at least three, four, five, six, seven,eight, nine, ten or eleven human interferon alpha (IFN-α) subtypesselected from the group consisting of IFN-αA, IFN-αB2, IFN-αC, IFN-αF,IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK, IFN-αWA and IFN-α4a with aK_(D) of about 5×10⁻¹⁰ M or less, about 1×10⁻¹⁰ M or less, about 5×10⁻¹¹M or less, about 1×10⁻¹¹ M or less, or about 5×10⁻¹² M or less.

The affinity of an antibody to IFN-ω or to various IFN-α subtypes may bedetermined experimentally using any suitable method. Such methods mayutilize ProteOn XPR36, Biacore 3000 or KinExA instrumentation, ELISA orcompetitive binding assays known to those skilled in the art. Themeasured affinity of a particular antibody/IFN-ω or antibody/IFN-αsubtypes interaction may vary if measured under different conditions(e.g., osmolarity, pH). Thus, measurements of affinity and other bindingparameters (e.g., K_(D), K_(on), K_(off)) are preferably made withstandardized conditions and a standardized buffer, such as the bufferdescribed herein. Skilled in the art will appreciate that the internalerror for affinity measurements for example using Biacore 3000 orProteOn (measured as standard deviation, SD) can typically be within5-33% for measurements within the typical limits of detection. Thereforethe term “about” reflects the typical standard deviation in the assay.For example, the typical SD for a K_(D) of 1×10⁻⁹ M is up to+0.33×10⁻⁹M.

The antibodies binding human IFN-ω and IFN-α subtypes with a desiredaffinity and neutralization profile may be selected from libraries ofvariants or fragments by panning with human IFN-ω and/or IFN-α subtypesand optionally by further antibody affinity maturation. In an exemplarypanning campaign, phage libraries may be panned sequentially or using amixture of chimpanzee IFN-ω) and human IFN-α subtypes IFN-α2, IFN-αI,IFN-αH2, IFN-αG and IFN-αF. Alternatively, antibodies of the inventionmay be generated by immunizing mice with chimpanzee and cynomolgusIFN-ω, human IFN-α subtypes IFN-αD, IFN-αJ1, IFN-αC, IFN-αB2, IFN-αH2,IFN-αA, IFN-α4a, IFN-αG, IFN-αF, IFN-αWA and IFN-αI, and screening thehybriomas for binding to IFN-ω) and various IFN-α subtypes, andsubsequently assessing the neutralization ability of the antibodiesusing methods described herein.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises heavy chain complementarity determining region(HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) amino acid sequences of SEQ IDNOs: 109, 114 and 121, respectfully, and light chain complementaritydetermining region (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) amino acidsequences of SEQ ID NOs: 118, 119 and 120.

Exemplary such antibodies are antibodies IFWM3308, IFWM3307, IFWM3410,IFWM3322, IFWM3385, IFWM3416, IFWM3310, IFWM3400, IFWM3321, IFWM3522,IFWM3524, IFWM3320, IFWM3304, IFWM3520, IFWM3399, IFWM3314, IFWM3331,IFWM3405, IFWM3442, IFWM3525, IFWM3423, IFWM3444 and IFWM3421. Theseantibodies neutralize human IFN-ω and at least three IFN-α subtypes withan IC₅₀ value of about 1×10⁻¹⁰ M or less, and comprise a consensus LCDR1(SEQ ID NO: 118), LCDR2 (SEQ ID NO: 119), LCDR3 (SEQ ID NO: 120), HCDR2(SEQ ID NO: 114) and HCDR3 (SEQ ID NO: 121) amino acid sequences and aconstant HCDR1 (SEQ ID NO: 109) amino acid sequence. Antibodies havingsubstitutions at least at VH residue position 103 of SEQ ID NOs: 28, 31,157 or 158, VL residue positions 30, 31, 32, 50, 91-94 or 96 of SEQ IDNOs: 35, 39, 40, 42, 46, 52, 53, 54, 71, 73, 75 or 135, and VL residuespositions 30, 31, 32, 50, 51, 92-95 or 97 of SEQ ID NOs: 57, 61, 62, 68and 150 resulted in antibodies having improved potency when compared tothe parental IFWM371 antibody.

SEQ ID NO: 118

QSIX₁X₂X₃X₄; wherein

X₁ is G, D, A, R, E, S, or N; X₂ is D, G, N, S, R, E or K; X₃ is F, A,N, T, S or V;

X₄ is Y, N or deleted.

SEQ ID NO: 119

X₅AS; wherein

X₅ is F, W or G. SEQ ID NO: 120

QQX₆X₇X₈X₉PX₁₀T; wherein

X₆ is A, G, S or W; X₇ is L, Y, H, W, F or I; X₈ is D or 5; X₉ is F, T,L, N or W; and X₁₀ is L, F or I. SEQ ID NO: 114

IX₁₁X₁₂SDSDT; wherein

X₁₁ is D or A; and X₁₂ is P or A. SEQ ID NO: 121

ARHPGLX₁₃WAPDFDY; wherein

X₁₃ is A or N. SEQ ID NO: 109 GYSFTSYW

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 114, 121,159, 119 and 160, respectively.

Exemplary such antibodies are antibodies IFWM3400, IFWM3321, IFWM3522,IFWM3524, IFWM3320, IFWM3304, IFWM3520, IFWM3399, IFWM3314, IFWM3331,IFWM3405, IFWM3442, IFWM3525, IFWM3423, IFWM3444 and IFWM3421. Theseantibodies neutralize human IFN-ω and at least six IFN-α subtypes withan IC₅₀ value of about 1×10⁻¹⁰ M or less, and comprise a consensus LCDR1(SEQ ID NO: 159), LCDR2 (SEQ ID NO: 119), LCDR3 (SEQ ID NO: 160), HCDR2(SEQ ID NO: 114) and HCDR3 (SEQ ID NO: 121) amino acid sequences and aconstant HCDR1 (SEQ ID NO: 109) amino acid sequence.

SEQ ID NO: 159

QSIX₁₄X₁₈X₁₆X₁₇; wherein

X₁₄ is G, D, A, E, S, or N; X₁₅ is D, G, N, S or R; X₁₆ is F, A, N, S orV; and

X₁₇ is Y, N or deleted.

SEQ ID NO: 160

QQX₁₈X₁₉X₂₀X₂₁PX₂₂T; wherein

X₁₈ is A, G or S; X₁₉ is Y, H, W or F; X₂₀ is D or S; X₂₁ is F, T, L orW; and X₂₂ is L, For I.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 114, 121,161, 119 and 162, respectively.

Exemplary such antibodies are antibodies IFWM3405, IFWM3442, IFWM3525,IFWM3423, IFWM3444 and IFWM3421. These antibodies neutralize human IFN-ωand at least ten IFN-α subtypes with an IC₅₀ value of at least about2×10⁻¹⁰ M or less, about 1.5×10⁻¹⁰ M or less, or about 1×10⁻¹⁰ M orless, and comprise a consensus LCDR1 (SEQ ID NO: 161), LCDR2 (SEQ ID NO:119), LCDR3 (SEQ ID NO: 162), HCDR2 (SEQ ID NO: 114) and HCDR3 (SEQ IDNO: 121) sequences and a constant HCDR1 (SEQ ID NO: 109) sequence.

SEQ ID NO: 161

QSIX₂₃X₂₄X₂₅X₂₆; wherein

X₂₃ is A or D; X₂₄ is N or G; X₂₅ is F, N or S; and

X₂₆ is Y, N or deleted.

SEQ ID NO: 162

QQX₂₇X₂₈X₂₉X₃₀PX₃₁T; wherein

X₂₇ is G or S; X₂₈ is Y; X₂₉ is D; X₃₀ is F, T or L; and X₃₁ is L, F orI.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention neutralizes human IFN-ω and at least ten human IFN-αsubtypes selected from the group consisting of IFN-αA, IFN-αB2, IFN-αC,IFN-αF, IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK, IFN-αWA and IFN-α4a.

In some embodiments of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, the antibody neutralizes human IFN-ω and at least the human IFN-αsubtypes IFN-αA, IFN-αB2, IFN-αC, IFN-αF, IFN-αG, IFN-αH2, IFN-αJ1 andIFN-α4a.

In some embodiments of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, the antibody does not bind or neutralize IFN-αD or IFN-α 1.

In some embodiments of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, the antibody does not bind or neutralize IFN-β.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises

-   -   the HCDR1 amino acid sequence of SEQ ID NO: 109;    -   the HCDR2 amino acid sequence of SEQ ID NOs: 111, 112 or 113;    -   the HCDR3 amino acid sequence of SEQ ID NOs: 115 or 116;    -   the LCDR1 amino acid sequence of SEQ ID NOs: 76, 77, 78, 79, 80,        81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91;    -   the LCDR2 amino acid sequence of SEQ ID NOs: 93, 94 or 95; and    -   the LCDR3 amino acid sequence of SEQ ID NOs: 96, 97, 98, 99,        100, 101, 102, 103, 104, 105, 106 or 107.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs:

-   -   a) 109, 113, 116, 77, 93 and 104, respectively;    -   b) 109, 113, 116, 85, 93 and 96, respectively;    -   c) 109, 113, 115, 79, 95 and 107, respectively;    -   d) 109, 113, 116, 76, 93 and 103, respectively;    -   e) 109, 113, 115, 85, 93 and 96, respectively;    -   f) 109, 113, 115, 89, 95 and 100, respectively;    -   g) 109, 113, 116, 86, 93 and 105, respectively;    -   h) 109, 113, 115, 76, 93 and 103, respectively;    -   i) 109, 113, 116, 80, 93 and 97, respectively;    -   j) 109, 113, 116, 84, 93 and 97, respectively;    -   k) 109, 113, 116, 90, 93 and 97, respectively;    -   l) 109, 113, 116, 88, 93 and 102, respectively;    -   m) 109, 113, 116, 87, 93 and 105, respectively;    -   n) 109, 113, 116, 91, 93 and 106, respectively;    -   o) 109, 113, 115, 80, 93 and 97, respectively;    -   p) 109, 113, 116, 83, 93 and 101, respectively;    -   q) 109, 113, 116, 82, 94 and 98, respectively;    -   r) 109, 113, 115, 78, 95 and 100, respectively;    -   s) 109, 111, 116, 81, 93 and 106, respectively;    -   t) 109, 113, 116, 82, 94 and 99, respectively;    -   u) 109, 113, 115, 81, 93 and 106, respectively;    -   v) 109, 112, 116, 81, 93 and 106, respectively; or    -   w) 109, 113, 116, 81, 93 and 106, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,77, 93 and 104, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,85, 93 and 96, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 115,79, 95 and 107, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,76, 93 and 103, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 115,85, 93 and 96, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 115,89, 95 and 100, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,86, 93 and 105, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 115,76, 93 and 103, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,80, 93 and 97, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,84, 93 and 97, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,90, 93 and 97, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,88, 93 and 102, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,87, 93 and 105, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,91, 93 and 106, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 115,80, 93 and 97, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,83, 93 and 101, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,82, 94 and 98, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 115,78, 95 and 100, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 111, 116,81, 93 and 106, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,82, 94 and 99, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 115,81, 93 and 106, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 112, 116,81, 93 and 106, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, theLCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109, 113, 116,81, 93 and 106, respectively.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodycomprises the VH and the VL wherein the VH comprises the amino acidsequence of SEQ ID NOs: 28, 31, 157 or 158.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodycomprises the VH and the VL, wherein the VL comprises the amino acidsequence of SEQ ID NOs: 35, 39, 40, 42, 46, 52, 53, 54, 57, 61, 62, 68,71, 73, 75, 135 or 150.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodycomprises the VH of SEQ ID NOs: 28, 31, 157 or 158, and the VL of SEQ IDNOs: 35, 39, 40, 42, 46, 52, 53, 54, 57, 61, 62, 68, 71, 73, 75, 135 or150.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH and the VL of SEQ ID NOs: 28 and 40, 28and 39, 31 and 62, 28 and 54, 31 and 39, 31 and 68, 28 and 42, 31 and54, 28 and 53, 28 and 73, 28 and 75, 28 and 52, 28 and 35, 28 and 135,31 and 53, 28 and 46, 28 and 61, 31 and 57, 157 and 71, 28 and 150, 31and 71, 158 and 71, or 28 and 71.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodycomprises the VH and the VL, wherein the VH comprises the amino acidsequence of SEQ ID NOs: 28, 30, 31, 157 or 158.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodycomprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH ofSEQ ID NOs: 28, 30, 31, 157 or 158, and the LCDR1, LCDR2 and LCDR3 aminoacid sequences of the VL of SEQ ID NOs: 29, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 74, 148, 149,150, 151, 152 or 153, wherein the CDRs are defined according to Kabat,Chothia and/or IMGT.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodycomprises the VH and the VL, wherein the VL comprises the amino acidsequence of SEQ ID NOs: 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 74, 148, 149, 150, 151, 152 or153.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibodycomprises the VH and the VL, wherein the VH comprises the amino acidsequence of SEQ ID NOs: 28, 30, 31, 157 or 158, and the VL comprises theamino acid sequence of SEQ ID NOs: 29, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 74, 148, 149, 150,151, 152 or 153.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:29.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:32.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:33.

In some embodiment described herein, and in some embodiments of each andevery one of the numbered embodiments listed below s, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:34.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:35.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:36.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:37.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:38.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:39.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:40.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:41.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:42.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:43.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:44.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:45.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:46.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:47.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:48.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:49.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:50.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:51.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:52.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:53.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:54.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:55.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:56.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:57.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:58.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:59.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:60.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:61.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:62.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:63.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:64.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:65.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:66.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:67.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:68.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:69.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:32.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:33.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:34.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:35.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:36.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:37.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:38.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:39.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:40.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:41.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:42.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:43.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:44.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:45.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:46.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:47.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:48.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:49.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:50.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:51.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:52.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:53.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:54.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:56.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:57.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:58.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:59.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:60.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:61.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:62.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:63.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:64.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:65.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:66.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:67.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:68.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:69.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:32.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:33.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:34.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:35.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:36.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:37.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:38.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:39.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:40.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:41.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:42.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:43.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:44.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:45.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:46.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:47.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:48.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:49.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:50.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:51.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:52.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:53.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:54.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:56.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:57.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:58.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:59.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:60.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:61.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:62.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:63.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:65.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:66.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:67.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:68.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:69.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:70.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:70.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO:70.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:71.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 31 and the VL of SEQ ID NO:71.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:123.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:124.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:125.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:126.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:127.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:128.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:129.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:130.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:131.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:132.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:133.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:134.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:135.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:136.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:137.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:138.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:139.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:140.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:141.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:73.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:142.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:143.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:74.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:75.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:144.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:145.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:146.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:147.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:148.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:149.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:150.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:151.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:152.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:153.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 157 and the VL of SEQ IDNO: 71.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention comprises the VH of SEQ ID NO: 158 and the VL of SEQ IDNO: 71.

Variants of the anti-IFN-ω/α antibodies of the invention comprising VHor VL amino acid sequences shown in Table 9, Table 13, Table 15, Table17, Table 19 and Table 21 are within the scope of the invention. Forexample, variants may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11amino acid substitutions in the VH and/or VL that do not adverselyaffect the antibody properties. In some embodiments, the sequenceidentity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%to a VH or the VL amino acid sequence of the invention. Percent identitycan be determined for example by pairwise alignment using the defaultsettings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen,Carslbad, Calif.). Exemplary modifications are for example conservativeamino acid substitutions in the antigen-binding site or in the frameworkwithout adversely altering the properties of the antibody. Conservativesubstitutions may also be made to improve antibody properties, forexample stability or affinity. Conservative substitutions are those thattake place within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. Alternatively, theamino acid repertoire can be grouped as (1) acidic (aspartate,glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic(glycine, alanine, valine, leucine, isoleucine, serine, threonine), withserine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan);(5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteineand methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co.,1981). Furthermore, any native residue in the polypeptide may also besubstituted with alanine, as has been previously described for alaninescanning mutagenesis (MacLennan et al (1998) Acta Physiol. Scand. Suppl.643:55-67; Sasaki et al (1998) Adv. Biophys. 35:1-24). Desired aminoacid substitutions may be determined by those skilled in the art at thetime such substitutions are desired. The resulting antibody variants maybe tested for their characteristics using assays described herein.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the anti-IFN-α/ωantibody of the invention comprises a heavy chain variable region (VH)amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to SEQ ID NO: 28 and a light chain variable region (VL)amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO: 71.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the anti-IFN-α/ωantibody of the invention comprises a heavy chain variable region (VH)amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to SEQ ID NO: 28 and a light chain variable region (VL)amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO: 150.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the anti-IFN-α/ωantibody of the invention comprises a heavy chain variable region (VH)amino acid sequence at least 95% identical to SEQ ID NO: 28 and a lightchain variable region (VL) amino acid sequences at least 95% identicalto SEQ ID NO: 71.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the anti-IFN-α/ωantibody of the invention comprises a heavy chain variable region (VH)amino acid sequence at least 95% identical to SEQ ID NO: 28 and a lightchain variable region (VL) amino acid sequences at least 95% identicalto SEQ ID NO: 150.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the anti-IFN-α/ωantibody of the invention comprises a heavy chain variable region (VH)amino acid sequence at least 97% identical to SEQ ID NO: 28 and a lightchain variable region (VL) amino acid sequences at least 97% identicalto SEQ ID NO: 71.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the anti-IFN-α/ωantibody of the invention comprises a heavy chain variable region (VH)amino acid sequence at least 97% identical to SEQ ID NO: 28 and a lightchain variable region (VL) amino acid sequences at least 97% identicalto SEQ ID NO: 150.

Amino acid substitutions may be done for example by PCR mutagenesis(U.S. Pat. No. 4,683,195). Alternatively, libraries of variants may begenerated using known methods, for example using random (NNK) ornon-random codons, for example DVK codons, which encode 11 amino acids(Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp) and screeningthe libraries for variants with desired properties.

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, one from a heavy chain and one from a light chain, askilled artisan will recognize that alternative embodiments may comprisesingle heavy or light chain variable regions. The single variable regioncan be used to screen for variable domains capable of forming atwo-domain specific antigen-binding fragment capable of, for example,binding to human IFN-ω or various human IFN-α subtypes. The screeningmay be accomplished by phage display screening methods using for examplehierarchical dual combinatorial approach disclosed in Int. Pat. Publ.No. WO92/01047. In this approach, an individual colony containing eithera H or L chain clone is used to infect a complete library of clonesencoding the other chain (L or H), and the resulting two-chain specificantigen-binding domain is selected in accordance with phage displaytechniques as described. Therefore, the individual VH and VL polypeptidechains are useful in identifying additional antibodies specificallybinding to human IFN-ω or various IFN-α subtypes using the methodsdisclosed in Int. Pat. Publ. No. WO92/01047.

Antibodies of the invention may be made using a variety of technologiesfor generating antibodies. For example, the hybridoma method of Kohlerand Milstein, Nature 256:495, 1975 may be used to generate monoclonalantibodies. In the hybridoma method, a mouse or other host animal, suchas a hamster, rat or monkey, is immunized with human IFN-ω and/orvarious IFN-α subtypes or fragments of these proteins, followed byfusion of spleen cells from immunized animals with myeloma cells usingstandard methods to form hybridoma cells (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)). Coloniesarising from single immortalized hybridoma cells are screened forproduction of antibodies with desired properties, such as specificity ofbinding, cross-reactivity or lack thereof, and affinity for the antigen.

Various host animals may be used to produce the IFN-α/ω antibodies ofthe invention. For example, Balb/c mice may be used to generate mouseanti-human IFN-α/o) antibodies. The antibodies made in Balb/c mice andother non-human animals may be humanized using various technologies togenerate more human-like sequences. Exemplary humanization techniquesincluding selection of human acceptor frameworks are known to skilled inthe art and include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting(U.S. Pat. No. 6,818,749), Resurfacing (Padlan, Mol Immunol 28:489-499,1991), Specificity Determining Residues Resurfacing (U.S. Pat. Publ. No.20100261620), human-adaptation (or human framework adaptation) (U.S.Pat. Publ. No. US2009/0118127), Superhumanization (U.S. Pat. No.7,709,226) and guided selection (Osbourn et al (2005) Methods 36:61-68,2005; U.S. Pat. No. 5,565,332).

Humanized antibodies may be further optimized to improve theirselectivity or affinity to a desired antigen by incorporating alteredframework support residues to preserve binding affinity (backmutations)by techniques such as those disclosed as described in Int. Pat. Publ.No. WO90/007861 and in Int. Pat. Publ. No. WO92/22653.

Transgenic mice carrying human immunoglobulin (Ig) loci in their genomemay be used to generate human antibodies against a target protein, andare described in for example Int. Pat. Publ. No. WO90/04036, U.S. Pat.No. 6,150,584, Int. Pat. Publ. No. WO99/45962, Int. Pat. Publ. No.WO02/066630, Int. Pat. Publ. No. WO02/43478, Lonberg et al (1994) Nature368:856-9; Green et al (1994) Nature Genet. 7:13-21; Green & Jakobovits(1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int. Rev.Immunol. 13:65-93; Bruggemann et al (1991) Eur. J. Immunol.21:1323-1326; Fishwild et al (1996) Nat. Biotechnol. 14:845-851; Mendezet al (1997) Nat. Genet. 15:146-156; Green (1999) Immunol. Methods231:11-23; Yang et al (1999) Cancer Res. 59:1236-1243; Brüggemann andTaussig (1997) Curr. Opin. Biotechnol. 8:455-458; Int. Pat. Publ. No.WO02/043478). The endogenous immunoglobulin loci in such mice may bedisrupted or deleted, and at least one complete or partial humanimmunoglobulin locus may be inserted into the mouse genome usinghomologous or non-homologous recombination, using transchromosomes, orusing minigenes. Companies such as Regeneron(http://_www_regeneron_com), Harbour Antibodies(http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc.(OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni(http://_www.trianni_com) and Ablexis (http://_www_ablexis_com) can beengaged to provide human antibodies directed against a selected antigenusing technology as described above.

Human antibodies may be selected from a phage display library, where thephage is engineered to express human immunoglobulins or portions thereofsuch as Fabs, single chain antibodies (scFv), or unpaired or pairedantibody variable regions (Knappik et al (2000) J. Mol. Biol. 296:57-86;Krebs et al (2001) J. Immunol. Meth. 254:67-84; Vaughan et al (1996)Nature Biotechnology 14:309-314; Sheets et al (1998) PITAS (USA)95:6157-6162; Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381; Markset al (1991) J. Mol. Biol. 222:581). The antibodies of the invention maybe isolated for example from phage display library expressing antibodyheavy and light chain variable regions as fusion proteins withbacteriophage pIX coat protein as described in Shi et al (2010)J. Mol.Biol. 397:385-96 and Int. Pat. Publ. No. WO09/085462). The libraries maybe screened for phage binding to human IFN-ω) and IFN-α and the obtainedpositive clones may be further characterized, the Fabs isolated from theclone lysates, and expressed as full length IgGs. Such phage displaymethods for isolating human antibodies are described in for example:U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.;U.S. Pat. Nos. 5,427,908 and 5, 580,717 to Dower et al.; U.S. Pat. Nos.5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 toGriffiths et al.

Preparation of immunogenic antigens and monoclonal antibody productionmay be performed using any suitable technique, such as recombinantprotein production. The immunogenic antigens may be administered to ananimal in the form of purified protein, or protein mixtures includingwhole cells or cell or tissue extracts, or the antigen may be formed denovo in the animal's body from nucleic acids encoding said antigen or aportion thereof.

In an exemplary method, phage display libraries may be panned againstbiotinylated human IFN-α2 or biotinylated human IFN-αG. After threerounds of panning, a polyclonal phage ELISA using human IFN-α2, IFN-αGand IFN-ω as antigens may be performed to detect the specific enrichmentof individual panning experiments. The phage demonstrating enrichmentfor binders to IFN-α2, IFN-αG and IFN-ω may be collected and furtherscreened in a standard ELISA assay for binding to additional IFN-αsubtypes in Fab format. The identified Fab clones may be cloned to fulllength antibodies and characterized further for their affinity andneutralization ability of human IFN-ω) and various IFN-α subtypes usingProteOn and ISRE reporter gene assay as described herein.

The antibodies of the invention may be human or humanized.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the IFN-α/ωantibodies of the invention comprise a VH framework derived from humangermline gene IGHV5-51 (SEQ ID NO: 155).

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the IFN-α/ωantibodies of the invention comprise a VL framework derived from humangermline gene IGKV1D-39 (SEQ ID NO: 156).

The antibodies of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, may be of IgA, IgD, IgE, IgG or IgM type. The antibodies of theinvention may be of IgG1, IgG2, IgG3, IgG4 type.

Immune effector properties of the antibodies of the invention may beenhanced or silenced through Fc modifications by techniques known tothose skilled in the art. For example, Fc effector functions such as C1qbinding, complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. can be providedand/or controlled by modifying residues in the Fc responsible for theseactivities. Pharmacokinetic properties of the antibodies of theinvention may be enhanced by mutating residues in the Fc domain thatextend antibody half-life (Strohl (2009) Curr Opin Biotechnol20:685-91). Exemplary Fc modifications are IgG4 S228P/L234A/L235A, IgG2M252Y/S254T/T256E (Dall'Acqua et al (2006) J. Biol. Chem. 281:23514-24;or IgG2 V234A/G237A/P238S, V234A/G237A/H268Q, H268AN309L/A330S/P331 orV234A/G237A/P238S/H268AN309L/A330S/P331S on IgG2 (Intl. Pat. Publ. No.WO11/066501), of those described in U.S. Pat. No. 6,737,056 (residuenumbering according to the EU numbering).

Additionally, antibodies of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, may be post-translationally modified by processes such asglycosylation, isomerization, deglycosylation or non-naturally occurringcovalent modification such as the addition of polyethylene glycolmoieties (pegylation) and lipidation. Such modifications may occur invivo or in vitro. For example, the antibodies of the invention may beconjugated to polyethylene glycol (PEGylated) to improve theirpharmacokinetic profiles. Conjugation may be carried out by techniquesknown to those skilled in the art. Conjugation of therapeutic antibodieswith PEG has been shown to enhance pharmacodynamics while notinterfering with function (Knigh et al (2004) Platelets 15:409-18; Leonget al (2001) Cytokine 16:106-19; Yang et al (2003) Protein Eng.16:761-70).

Antibodies or fragments thereof of the invention modified to improvestability, selectivity, cross-reactivity, affinity, immunogenicity orother desirable biological or biophysical property are within the scopeof the invention. Stability of an antibody is influenced by a number offactors, including (1) core packing of individual domains that affectstheir intrinsic stability, (2) protein/protein interface interactionsthat have impact upon the HC and LC pairing, (3) burial of polar andcharged residues, (4) H-bonding network for polar and charged residues;and (5) surface charge and polar residue distribution among other intra-and inter-molecular forces (Worn et al (2001) J. Mol. Biol.305:989-1010). Potential structure destabilizing residues may beidentified based upon the crystal structure of the antibody or bymolecular modeling in certain cases, and the effect of the residues onantibody stability can be tested by generating and evaluating variantsharboring mutations in the identified residues. One of the ways toincrease antibody stability is to raise the thermal transition midpoint(T_(m)) as measured by differential scanning calorimetry (DSC). Ingeneral, the protein T_(m) is correlated with its stability andinversely correlated with its susceptibility to unfolding anddenaturation in solution and the degradation processes that depend onthe tendency of the protein to unfold (Remmele et al (2000) Biopharm13:36-46,). A number of studies have found correlation between theranking of the physical stability of formulations measured as thermalstability by DSC and physical stability measured by other methods (Guptaet al (2003) AAPS PharmSci 5E8; Zhang et al (2004) J. Pharm. Sci.93:3076-89; Maa et al (1996) Int. J. Pharm. 140:155-68; Bedu-Addo et al(2004) Pharm. Res. 21:1353-61; Remmele et al (1997) Pharm. Res.15:200-8). Formulation studies suggest that a Fab T_(m) has implicationfor long-term physical stability of a corresponding mAb. Differences inamino acids in either framework or within the CDRs could havesignificant effects on the thermal stability of the Fab domain (Yasui etal (1994) FEBS Lett. 353:143-6).

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention competes with binding to the human IFN-ω) with an isolatedantibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 71.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention competes with binding to the human IFN-ω) with an isolatedantibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:150.

Competition between specific binding to human IFN-ω with antibodies ofthe invention comprising certain VH and VL sequences may be assayed invitro using well known methods. For example, binding of MSD Sulfo-Tag™NHS-ester-labeled antibody to human to human IFN-ω in the presence of anunlabeled antibody can be assessed by ELISA, or Bioacore analyses orflow cytometry may be used to demonstrate competition with theantibodies of the current invention.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention binds to and neutralizes a biological activity of a humaninterferon omega (IFN-ω) and at least three, four, five, six, seven,eight, nine, ten or eleven human interferon alpha (IFN-α) subtypes,wherein the antibody binds IFN-ω of SEQ ID NO: 1 at least at residuesF27, L30 and R33 of.

The residues F27, L30 and R33 IFN-ω define a minimal epitope requiredfor broad neutralizing activity of the IFN-α/ω antibodies of theinvention. Crystal structure of several antibody/IFN-α or antibody/IFN-ωcomplexes revealed the three residues provide predominant contributionsto antibody binding. The F27 residue is conserved in all human IFN-αsexcept IFN-αD (α1), to which antibodies of the invention do not bind.Both L30 and R33 are conserved in all human IFN-αs as well as in humanIFN-ω. Further confirmation of the contribution of F27 to the epitope isevident from the binding studies with various cyno IFN-α subtypes: theantibodies of the invention do not bind cyno IFN-α13, which, like humanIFN-αD, has a serine at position 27 (S27).

In another embodiment described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention binds human IFN-ω of SEQ ID NO: 1 at least at residuesS25, P26, F27, L28, L30, K31, R33, R34 and D35.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention binds to and neutralizes a biological activity of a humaninterferon omega (IFN-ω) and at least three, four, five, six, seven,eight, nine, ten or eleven human interferon alpha (IFN-α) subtypes,wherein the antibody binds human IFN-ω of SEQ ID NO: 1 at one or moreresidues including F27.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, the antibody ofthe invention is a bispecific antibody that binds to and neutralizes abiological activity of a human interferon omega (IFN-ω) and at leastthree, four, five, six, seven, eight, nine, ten or eleven humaninterferon alpha (IFN-α) subtypes and binds BLyS, CD40L, IL-6, CD27,BDCA2, IL-12, IL-23, IFN-αD, IL-17, CD20, IL-10, CD22, IL-21, ICOS,ICOSL or IFN-γ.

Given the presence of elevated IFN-ω) in SLE patients, and thedemonstration that IFN-ω) can induce BLyS secretion in PBMCs in vitro,combined blockade of IFN-α/ω in SLE patients may be more effective atreducing BLyS levels in comparison to anti IFN-α specific approaches.The extent of IFN-signature and IFN activity in SLE patients appears tocorrelate with soluble BLyS levels.

The IFN-α/ω antibodies of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, may be engineered into bispecific antibodies which are alsoencompassed within the scope of the invention. The VL and/or the VHregions of the antibodies of the invention may be engineered usingpublished methods into single chain bispecific antibodies as structuressuch as TandAb® designs (Int. Pat. Publ. No. WO99/57150; U.S. Pat. Publ.No. US2011/0206672) or into bispecific scFVs as structures such as thosedisclosed in U.S. Pat. No. 5,869,620; Int. Pat. Publ. No. WO95/15388,lnt. Pat. Publ. No. WO97/14719 or Int. Pat. Publ. No WO11/036460.

The VL and/or the VH regions of the antibodies of the invention may beengineered into bispecific full length antibodies, where each antibodyarm binds a distinct antigen or epitope. Such bispecific antibodies aretypically made by modulating the CH3 interactions between the twoantibody heavy chains to form bispecific antibodies using technologiessuch as those described in U.S. Pat. No. 7,695,936; Int. Pat. Publ. No.WO04/111233; U.S. Pat. Publ. No. 2010/0015133; U.S. Pat. Publ. No.2007/0287170; Int. Pat. Publ. No. WO2008/119353; U.S. Pat. Publ. No.2009/0182127; U.S. Pat. Publ. No. 2010/0286374; U.S. Pat. Publ. No.2011/0123532; Int. Pat. Publ. No. WO2011/131746; Int. Pat. Publ. No.WO2011/143545; or U.S. Pat. Publ. No. 2012/0149876.

For example, bispecific antibodies of the invention may be generated invitro in a cell-free environment by introducing asymmetrical mutationsin the CH3 regions of two monospecific homodimeric antibodies andforming the bispecific heterodimeric antibody from two parentmonospecific homodimeric antibodies in reducing conditions to allowdisulfide bond isomerization according to methods described in Intl.Pat. Publ. No. WO2011/131746. In the methods, the first monospecificbivalent antibody (e.g., anti-IFN-α/ω antibody of the invention) and thesecond monospecific bivalent antibody (e.g., anti-BLyS, anti-CD40L,anti-IL-6, anti-CD27, anti-BDCA2, anti-IL-12, anti-IL-23, anti-IFN-αD,anti-IL-17, anti-CD20, anti-IL-10, anti-CD22, anti-IL-21, anti-ICOS,anti-ICOSL or anti-IFN-γ antibody) are engineered to have certainsubstitutions at the CH3 domain that promote heterodimer stability; theantibodies are incubated together under reducing conditions sufficientto allow the cysteines in the hinge region to undergo disulfide bondisomerization; thereby generating the bispecific antibody by Fab armexchange. The incubation conditions may optimally be restored tonon-reducing. Exemplary reducing agents that may be used are2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol(DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine andbeta-mercaptoethanol, preferably a reducing agent selected from thegroup consisting of: 2-mercaptoethylamine, dithiothreitol andtris(2-carboxyethyl)phosphine. For example, incubation for at least 90min at a temperature of at least 20° C. in the presence of at least 25mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH offrom 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

Exemplary CH3 mutations that may be used in a first heavy chain and in asecond heavy chain of the bispecific antibody are K409R and/or F405L.

Additional bispecific structures into which the VL and/or the VH regionsof the antibodies of the invention may be incorporated are for exampleDual Variable Domain Immunoglobulins (DVD) (Int. Pat. Publ. No.WO2009/134776), or structures that include various dimerization domainsto connect the two antibody arms with different specificity, such asleucine zipper or collagen dimerization domains (Int. Pat. Publ. No.WO2012/022811, U.S. Pat. Nos. 5,932,448; 6,833,441). DVDs are fulllength antibodies comprising the heavy chain having a structureVH1-linker-VH2-CH and the light chain having the structureVL1-linker-VL2-CL; linker being optional.

The VH and the VL binding BLyS, CD40L, IL-6, CD27, BDCA2, IL-12, IL-23,IFN-αD, IL-17, CD20, IL-10, CD22, IL-21, ICOS, ICOSL or IFN-γ to beincorporated into bispecific anti-IFN-α/ω antibodies may be generated denovo using methods described herein, or may be engineered from existingmonospecific antibodies. Exemplary anti-BLyS antibody that may be usedto generate the bispecific antibodies of the invention is BENLYSTA®.Exemplary CD40L antibodies that may be used are those described in U.S.Pat. Nos. 5,474,771, 5,747,037, Int. Pat. Publ. No. WO01/68860, Int.Pat. Publ. No. WO06/033702 or Int. Pat. Publ. No. WO08/118356. Exemplaryanti-IL-6 antibodies that may be used are those described in Int. Pat.Publ. No. WO06/119115, Int. Pat. Publ. No. WO10/056948, Int. Pat. Publ.No. WO10/088444 or Int. Pat. Publ. No. WO07/076927. Exemplary anti-CD27antibodies that may be used are those described in Int. Pat. Publ. No.WO13/138586, Int. Pat. Publ. No. WO11/130434 or Int. Pat. Publ. No.WO12/004367. Exemplary IL-12 and IL-23 antibody that may be used areSTELARA® Exemplary IL-23 antibodies that may be used are those describedin Int. Pat. Publ. No. WO07/005955, Int. Pat. Publ. No. WO07/027714,Int. Pat. Publ. No. WO08/103432, Int. Pat. Publ. No. WO07/106769, Int.Pat. Publ. No. WO07/147019 or Int. Pat. Publ. No. WO08/134659. ExemplaryIL-17 antibodies that may be used are those described in Int. Pat. Publ.No. WO06/013107, Int. Pat. Publ. No. WO06/054059 Int. Pat. Publ. No.WO07/070750, Int. Pat. Publ. No. WO08/134659, Int. Pat. Publ. No.WO07/149032, Int. Pat. Publ. No. WO08/021156, Int. Pat. Publ. No.WO08/047134, Int. Pat. Publ. No. WO09/130459, Int. Pat. Publ. No.WO10/025400, Int. Pat. Publ. No. WO11/053763 and Int. Pat. Publ. No.WO12/095662.

Another embodiment of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, is an antibody that binds to and neutralizes a biologicalactivity of a human interferon omega (IFN-ω) and at least three, four,five, six, seven, eight, nine, ten or eleven human interferon alpha(IFN-α) subtypes having certain VH and VL sequences, wherein theantibody VH is encoded by a first polynucleotide and the antibody VL isencoded by a second synthetic polynucleotide. The polynucleotide may bea complementary deoxynucleic acid (cDNA), and may be codon optimized forexpression in suitable host. Codon optimization is a well-knowntechnology.

In some embodiments described herein, and in some embodiments of eachand every one of the numbered embodiments listed below, thepolynucleotides encoding the antibody VH or VL of the invention comprisethe sequences of SEQ ID NOs: 72, 92, 108, 110, 117 or 122.

Another embodiment of the invention is an isolated polynucleotideencoding any of the antibody heavy chain variable regions and/or theantibody light chain variable regions of the invention. Certainexemplary polynucleotides are disclosed herein, however, otherpolynucleotides which, given the degeneracy of the genetic code or codonpreferences in a given expression system, encode the antibodies of theinvention are also within the scope of the invention. Exemplarypolynucleotides are for example polynucleotides having the sequencesshown in SEQ ID NOs: 72, 92, 108, 110, 117 or 122. The polynucleotidesequences encoding a VH or a VL or a fragment thereof of the antibody ofthe invention may be operably linked to one or more regulatory elements,such as a promoter or enhancer, that allow expression of the nucleotidesequence in the intended host cell. The polynucleotide may be a cDNA.

Another embodiment of the invention is a vector comprising thepolynucleotide of the invention. Such vectors may be plasmid vectors,viral vectors, vectors for baculovirus expression, transposon basedvectors or any other vector suitable for introduction of the syntheticpolynucleotide of the invention into a given organism or geneticbackground by any means. For example, polynucleotides encoding lightand/or heavy chain variable regions of the antibodies of the invention,optionally linked to constant regions, are inserted into expressionvectors. The light and/or heavy chains may be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains may be operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Such control sequences include signal sequences, promoters (e.g.naturally associated or heterologous promoters), enhancer elements, andtranscription termination sequences, and are chosen to be compatiblewith the host cell chosen to express the antibody. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the proteinsencoded by the incorporated polynucleotides.

Suitable expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markerssuch as ampicillin-resistance, hygromycin-resistance, tetracyclineresistance, kanamycin resistance or neomycin resistance to permitdetection of those cells transformed with the desired DNA sequences.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, exemplary promoters include lad, lacZ,T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell,exemplary promoters include light and/or heavy chain immunoglobulin genepromoter and enhancer elements; cytomegalovirus immediate earlypromoter; herpes simplex virus thymidine kinase promoter; early and lateSV40 promoters; promoter present in long terminal repeats from aretrovirus; mouse metallothionein-I promoter; and various art-knowntissue specific promoters. For expression in a yeast cell, an exemplarypromoter is constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PH05 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following vectors are provided byway of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif.,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG(Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Another embodiment of the invention is a host cell comprising one ormore vectors of the invention. The term “host cell” refers to a cellinto which a vector has been introduced. It is understood that the termhost cell is intended to refer not only to the particular subject cellbut to the progeny of such a cell, and also to a stable cell linegenerated from the particular subject cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not be identical to theparent cell, but are still included within the scope of the term “hostcell” as used herein. Such host cells may be eukaryotic cells,prokaryotic cells, plant cells or archeal cells.

Escherichia coli, bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species are examples of prokaryotic host cells. Othermicrobes, such as yeast, are also useful for expression. Saccharomyces(e.g., S. cerevisiae) and Pichia are examples of suitable yeast hostcells Exemplary eukaryotic cells may be of mammalian, insect, avian orother animal origins. Mammalian eukaryotic cells include immortalizedcell lines such as hybridomas or myeloma cell lines such as SP2/0(American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NSO(European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK,ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murinecell lines. An exemplary human myeloma cell line is U266 (ATTCCRL-TIB-196). Other useful cell lines include those derived from ChineseHamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics,Walkersville, Md.), CHO-K1 (ATCC CRL-61) or DG44.

Another embodiment of the invention is a method of producing an antibodyof the invention comprising culturing the host cell of the invention inconditions that the antibody is expressed, and recovering the antibodyproduced by the host cell. Methods of making antibodies and purifyingthem are well known in the art. Once synthesized (either chemically orrecombinantly), the whole antibodies, their dimers, individual lightand/or heavy chains, or other antibody fragments such as VH and/or VL,may be purified according to standard procedures, including ammoniumsulfate precipitation, affinity columns, column chromatography, highperformance liquid chromatography (HPLC) purification, gelelectrophoresis, and the like (see generally Scopes, ProteinPurification (Springer-Verlag, N.Y., (1982)). A subject antibody may besubstantially pure, e.g., at least about 80% to 85% pure, at least about85% to 90% pure, at least about 90% to 95% pure, or at least about 98%to 99%, or more, pure, e.g., free from contaminants such as cell debris,macromolecules, etc. other than the subject antibody.

Another embodiment of the invention is a method for producing anantibody that binds to and neutralizes a biological activity of a humaninterferon omega (IFN-ω) and at least three, four, five, six, seven,eight, nine, ten or eleven human interferon alpha (IFN-α) comprising:

-   -   incorporating the first polynucleotide encoding the VH of the        antibody and the second polynucleotide encoding the VL of the        antibody into an expression vector;    -   transforming a host cell with the expression vector;    -   culturing the host cell in culture medium under conditions        wherein the VL and the VH are expressed and form the antibody;        and    -   recovering the antibody from the host cell or culture medium.

The polynucleotides encoding certain VH or VL sequences of the inventionare incorporated into vectors using standard molecular biology methods.Host cell transformation, culture, antibody expression and purificationare done using well known methods.

Methods of Treatment

IFN-α/ω antibodies of the invention may be utilized to treatimmune-mediated inflammatory diseases or autoimmune diseases such aslupus, including systemic lupus erythematosus (SLE) or cutaneous lupuserythematosus (CLE), or other immune-mediated inflammatory diseases suchas psoriasis, immune thrombocytopenia (ITP), Aicardi-Goutieres syndrome(AGS), systemic sclerosis, Sjögren's syndrome, myositis, common variableimmune deficiency (CVID), autoimmune thyroid disease, type I diabetes,rheumatoid arthritis, transplant rejection or graft versus host disease(GVHD). These diseases may be associated with increased production ofIFN-α and/or IFN-ω or type I IFN signature.

One embodiment of the invention is a method of treating animmune-mediated inflammatory disease or an autoimmune disease,comprising administering a therapeutically effective amount of anisolated antibody that binds to and neutralizes a biological activity ofa human interferon omega (IFN-ω) and at least three, four, five, six,seven, eight, nine, ten or eleven human interferon alpha (IFN-α)subtypes to a patient in need thereof for a time sufficient to treat theimmune-mediated inflammatory disease or autoimmune disease.

Another embodiment of the invention is a method of treating lupus,comprising administering a therapeutically effective amount of anisolated antibody that binds to and neutralizes a biological activity ofa human interferon omega (IFN-ω) and at least three, four, five, six,seven, eight, nine, ten or eleven human interferon alpha (IFN-α)subtypes to a patient in need thereof for a time sufficient to treatlupus.

In some embodiments, lupus is systemic lupus erythematosus (SLE) orcutaneous lupus erythematosus (CLE).

In some embodiments, the patient has lupus nephritis.

In some embodiments, the immune-mediated inflammatory disease or theautoimmune disease is psoriasis, immune thrombocytopenia (ITP),Aicardi-Goutieres syndrome (AGS), systemic sclerosis, Sjögren'ssyndrome, myositis, common variable immune deficiency (CVID), autoimmunethyroid disease, type I diabetes, rheumatoid arthritis, transplantrejection or graft versus host disease (GVHD).

Another embodiment of the invention is a method of treating a chronicviral infection, comprising administering a therapeutically effectiveamount of an isolated antibody that binds to and neutralizes abiological activity of a human interferon omega (IFN-ω) and at leastthree, four, five, six, seven, eight, nine, ten or eleven humaninterferon alpha (IFN-α) subtypes to a patient in need thereof for atime sufficient to treat the chronic viral infection.

IFN-I is well known to have a protective role in acute viral infection.Recently, IFN-I has been demonstrated to have an immunosuppressive rolein chronic viral infections through a mechanism at least partiallymediated by IL-10 and programmed cell death 1 ligand 1 (PDL1) (Teijaroet al., Science 340, 207-211, (2013); Wilson et al., Science 340,202-207, 2013). Combined blockade of multiple IFN-α subtypes and IFN-ωmay offer beneficial effects in patients with chronic viral infectionsincluding HIV and hepatitis C by down-modulating an immunosuppressiveenvironment conducive to viral persistence.

In some embodiments, the chronic viral infection is HIV or hepatitis C.

“Treatment” or “treat” refers to therapeutic treatment. Patients thatmay be treated also include those prone to or susceptible to have thedisorder, of those in which the disorder is to be prevented. Individualsin need of treatment include those already with the disorder or asymptom of the disorder. Beneficial or desired clinical results includealleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

Exemplary antibodies that may be used in the methods of the inventioncomprise VH, VL, HCDR and/or LCDR regions as shown in tables 9, 13, 15,17, 19, 21, 22, 23, 24, 25, 26 or 27, and antibodies IFWM3308, IFWM3307,IFWM3410, IFWM3322, IFWM3385, IFWM3416, IFWM3310, IFWM3400, IFWM3321,IFWM3522, IFWM3524, IFWM3320, IFWM3304, IFWM3520, IFWM3399, IFWM3314,IFWM3331, IFWM3405, IFWM3442, IFWM3525, IFWM3423, IFWM3444 and IFWM3421.

Other exemplary antibodies that may be used in the methods of theinvention described herein, and in some embodiments of each and everyone of the numbered embodiments listed below are antibodies that bind toand neutralize a biological activity of a human interferon omega (IFN-ω)and at least three, four, five, six, seven, eight, nine, ten or elevenhuman interferon alpha (IFN-α) subtypes, wherein the antibody bindsIFN-ω) of SEQ ID NO: 1 at least at residues F27, L30 and R33.

Other exemplary antibodies that may be used in the methods of theinvention described herein, and in some embodiments of each and everyone of the numbered embodiments listed below, are antibodies that bindhuman IFN-ω) of SEQ ID NO: 1 at least at residues S25, P26, F27, L28,L30, K31, R33, R34 and D35.

The methods of the invention may be used to treat an animal patientbelonging to any classification. Examples of such animals includemammals such as humans, rodents, dogs, cats and farm animals.

The antibodies of the invention may be useful in the preparation of amedicament for such treatment, wherein the medicament is prepared foradministration in dosages defined herein. SLE is a chronic multiorganautoimmune disease with both genetic and environmental factorcontributing to its development.

SLE is characterized by production of pathogenic autoantibodies andtissue deposition of immune complexes, resulting in tissue damage acrossmultiple organs. Combinations of cutaneous, musculoskeletal,hematological, neurological and renal complications are seen inpatients, with periods of flare-ups and remissions. Lupus nephritis isdefined as a case of SLE with a diagnosis of nephritis, proteinuria,hematuria and/or renal failure. In lupus nephritis patients, renalinvolvement is characterized by proteinuria (>0.5 g/24 hours), and/orred blood cells or casts in urine specimens.

Not wishing to be bound by any particular theory, it is suggested thatSLE triggers, such autoantibody immune complexes, invoke type I IFNresponses associated with overproduction of IFN-α and IFN-ω, but notIFN-β. Therefore, IFN-α/ω antibodies of the invention may provide a moreefficacious treatment of lupus and other immune-mediated inflammatorydisease, broadly inhibiting IFN-ω and multiple IFN-α subtypes whilesparing IFN-β function, which may play a more critical role in antiviraldefense and which molecule may have no biological releavance in lupus.For example, anti-IFN-β antibodies failed to neutralize patient serumactivity from both SLE and AGS patients, a disease also associated withelevated type IFN-I activity and IFN signature (Hooks et al., Arthritisand Rheumatism 25:396-400, 1982; Hua et al., Arthritis and Rheumatism54: 1906 (June, 2006); Rice et al., Lancet Neurologydoi:10.1016/S1474-4422(13)70258-8 (2013)).

Other types of lupus in addition to SLE include cutaneous lupuserythematosus (CLE) and pediatric lupus.

Symptoms associated with lupus include joint pain and stiffness,nonerosive arthritis, muscle aches, pains, weakness, fever, malaise,ulcers on oral tissues, cutaneous manifestations (e.g., butterfly-shapedrash across the nose and cheeks; sunlight-induced dermatologicalflares), unusual weight loss or weight gain, anemia, low lymphocyteand/or platelet counts, neurological or neuropsychiatric manifestations(e.g., trouble thinking, memory problems, confusion, depression,headache, seizures, strokes), kidney problems (e.g., nephritis, e.g.,glomerulonephritis), sun or light sensitivity, hair loss, purple or palefingers from stress or cold, vascular lesions or other vascularmanifestations, or cardio-pulmonary symptoms such as pericarditis orpleuritis. Elevated levels of interleukins IL-1, IL-6, IL-10, I1-12,IL-17, IL-18, IL-5 and IL-16; TNF-α or Type I interferons, as well asoverexpression of IFN inducible genes is documented in lupus patients.Patients may have elevated levels of autoantibodies against nuclear andcellular components such as double stranded DNA (dsDNA),ribonucleoprotein (RNP), SS-a/Ro, SS-b/La, phospholipids, histones orcardiolipin. Patients may have immune complex deposition in at least onetissue.

SLE may be diagnosed or classified for example using recommendations bythe American College of Rheumatology (ACR), or by the Systemic LupusInternational Collaborating Clinics Criteria (SLICC) for theClassification of Systemic Lupus Erythematosus. For example, the 2012SLICC criteria require that patients demonstrate at least 4 of 11criteria, with at least one clinical and one immunologic criterion, orlupus nephritis verified with biopsy in the presence of anti-DNAantibodies (ADA) or anti-nucleic acid antibodies (ANA). Clinicalcriteria are acute cutaneous lupus, chronic cutaneous lupus, oral ornasal ulcers, non-scarring alopecia, arthritis, serositis, renalsymptoms, neurologic symptoms, hemolytic anemia, leukopenia orthrombocytopenia (<100,000/mm³). Immunologic criteria include ANA, ADA,anti-Sm, anti-phospholipid antibodies, low complement (C3, C4 or CH50)or direct Coombs' test, which does not count in the presence ofhemolytic anemia (Petre et al., Arthritis and Rheumatism August 2012).Active disease may be defined by one British Isles Lupus ActivityGroup's (BILAG) “A” criteria or two BILAG “B” criteria; SLE DiseaseActivity Index (SLEDAI); or systemic lupus erythematosus (SLE) responderindex (SRI) described in Furie et al., Arthritis Rheum. 61(9): 1143-51(2009).

SLE severity and disease activity may be defined by a BILAG score by aclinician with expertise in SLE. The BILAG 2004 index is used todetermine the BILAG score (see Yee, et al. Arthritis & Rheumatism54:3300-3305, 2006; Isenberg et al., Rheumatology 44:902-906; 2005). TheBILAG 2004 index assesses 97 clinical signs, symptoms, and laboratoryparameters across nine organ system domains: constitutional,mucocutaneous, neuropsychiatric, musculoskeletal, cardiorespiratory,gastrointestinal, ophthalmic, renal, and hematological. The 97 symptomsare rated with respect to severity over the previous month (4 weeks) andwith respect to any change from the previous examination (new,improving, stable, worsening, absent). A single alphabetic score (Athrough E) for each of the nine domains is then derived from theexamination results in each organ category. Table 2 shows the BILAGcategories.

TABLE 2 Category Definition A Severe disease activity requiring any ofthe following treatment: 1. Systemic high dose oral glucocorticoids(equivalent to prednisolone >20 mg/day); 2. Intravenous pulseglucocorticoids (equivalent to pulse methylprednisolone ≥500 mg); 3.Systemic immunomodulators (include biologicals, immunoglobulins andplasmapheresis); 4. Therapeutic high dose anticoagulation in thepresence of high dose steroids or immunomodulators, e.g., warfarin withtarget INR 3-4. B Moderate disease activity requiring any of thefollowing treatment: 1. Systemic low dose oral glucocorticoids(equivalent to prednisolone ≤20 mg/day); 2. Intramuscular orintra-articular or soft tissue glucocorticoids injection (equivalent tomethylprednisolone <500 mg). C Stable mild disease. D Inactive diseasebut previously affected. E System never involved.

CLE is further classified to acute (ACLE), subacute (SCLE), chronic(CCLE) or intermittent (ICLE) CLE depending on the constellation ofclinical features and duration of the cutaneous lesions, laboratoryabnormalities, and skin biopsy histological changes. Classification andclinical manifestations of the various CLE forms are reviewed in Kuhnand Landmann, J Autiommunity 48-49:14-19, 2014.

A type I IFN gene signature has been reported to positively correlatewith both clinical and serological features of lupus (Karageorgas etal., J Biomed Biotechnol 273907, 2011 Baechler et al., Proc Natl AcadSci USA 100:2610-2615, 2003, Bennett et al., J Exp Med 197:711-723,2003, Dall′era et al. Ann Rheum Dis 64: 1692-1697, 2005, Niewold et al.Genes Immun 8: 492-502, 2007).). A preponderance of autoantibodies inconjunction with their impaired clearance leads to a feedback cycle ofIFN production where Fc receptor-dependent internalization of immunecomplexes into plasmacytoid dendritic cells (pDC) leads to increasedamounts of IFN and thus establishment of the IFN signature. In clinicaltrials, anti-IFN-α antibodies in SLE patients have demonstrated partialreduction of the type I IFN signature in the majority of patientsexhibiting the IFN signature and slight efficacy in exploratory analysis(Petri et al., Arthritis and rheumatism 65, 1011 (April, 2013); MerrillJ et al., Annals of the rheumatic diseases 70, 314 (2011); Kennedy etal., The 10th International Congress on SLE, Buenos Aires, ArgentinaOral Presentation 5, 022, (Apr. 20, 2013)).

The standard of care in lupus management is based on current, acceptedmedical practice patterns, approved guidance documents developed byrheumatology societies (e.g. American College of Rheumatology, EuropeanLeague Against Rheumatism) and the discretion of treating physicians.Lupus patients continue to have disease activity long after thediagnosis is made, even with proper management, often involving neworgan systems or specific organ system damage. There are three patternsof disease activity in lupus: the flare (or remitting, relapsing diseaseactivity), chronically active disease, and long quiescence. Thesedisease patterns are characterized using systematic clinicalassessments, routine laboratory tests, standardized measures of diseaseactivity, and integration of these assessments with the patient's ownperceptions of health status and quality of life. As the patient's signsand symptoms of flare persist or worsen, the physician may find that achange in medications and/or dosages is warranted. The medications usedto control lupus include, but is not limited to the following: (1)NSAIDs, including over-the-counter NSAIDs, e.g., naproxen (Aleve) andibuprofen (Advil, Motrin, others), and stronger NSAIDs available byprescription; (2) Antimalarial drugs, e.g., hydroxychloroquine(Plaquenil); (3) Corticosteroids., e.g., Prednisone and other types ofcorticosteroids, and (4) Immune suppressants, e.g., cyclophosphamide(Cytoxan), azathioprine (Imuran, Azasan), mycophenolate (Cellcept),leflunomide (Arava) and methotrexate (Trexall).

The antibodies of the invention may be tested for their efficacy invitro in disease relevant cells using disease relevant IFN preparations.Such in vitro testing may be for example evaluation of inhibition of IFNproduction induced by SLE patient immune complexes in whole blood, orassessment of ability of the antibodies to reduce the IFN signature inwhole blood as described herein. Animal models of lupus may also beused, such as NZB/NZW F1 mice that exhibit a time-dependent andfemale-biased disease with several features of human lupus includingglomerulonephritis. However, as mice do not produce IFN-ω) theirutilization as a model to assess efficacy of the antibodies of theinvention is more limited.

In some embodiments, the patient exhibits a Type I interferon signature.“Type I interferon signature” or “interferon signature” as used hereinrefers to the upregulation of a subset of genes that are induced byIFN-I. Various type I IFN signatures are known, ranging from 3-27 genes.These signatures may be utilized for example as pharmacodynamics markersto assess target engagement of Type I IFN inhibitors for treatment ofSLE and for purpose of SLE patient stratification.

An exemplary Type I interferon signature is shown in Table 3, consistingof 21 upreguated genes as described in Yao et al., Arthritis andrheumatism 60, 1785 (June, 2009). Other exemplary type I interferonsignatures are described in Tcherepanova, I., et al., Annals of therheumatic diseases 71(Suppl3) (2012) and Richardson, B. et al.Development of A Quantitative PCR Method to Determine InterferonSignature Metric Status in SLE Patients: Distribution and Clinical &Serological Associations in Two Lupus Clinical Trials. ACR/ARHP 2012Annual Meeting Abstract 620 (2012).

In some methods, the anti-IFN-α/ω antibody is a bispecific antibody.

In some methods, the anti-IFN-α/ω bispecific antibody neutralizes BLyS,CD40L, IL-6, CD27, BDCA2, IL-12, IL-23, IFN-αD, IL-17 or CD20.

TABLE 3 Gene Number Symbol Gene Name 1 IFI27 interferon, alpha-inducibleprotein 27 2 IFI6 interferon, alpha-inducible protein 6 3 RSAD2 radicalS-adenosyl methionine domain containing 2 4 IFI44 Interferon-inducedprotein 44 5 IFI44L IFI44L interferon-induced protein 44-like 6 USP18ubiquitin specific peptidase 18 7 LY6E lymphocyte antigen 6 complex,locus E 8 OAS1 2',5'-oligoadenylate synthetase 1, 40/46 kDa 9 SIGLEC1SIGLEC1 sialic acid binding Ig-like lectin 1 10 ISG15 ISG15ubiquitin-like modifier 11 IFIT1 interferon-induced protein withtetratricopeptide repeats 12 OAS3 OAS3 2'-5'-oligoadenylate synthetase3, 100 kDa 13 HERC5 hect domain and RLD 5 14 MX1 myxovirus (influenzavirus) resistance 1 15 LAMP3 lysosomal-associated membrane protein 3 16EPSTI1 epithelial stromal interaction 1 (breast) 17 IFIT3interferon-induced protein with tetratricopeptide repeats 18 OAS22'-5'-oligoadenylate synthetase 2, 69/71 kDa 19 RTP4 receptor(chemosensory) transporter protein 4 20 PLSCR1 Phospholipid scramblase 121 DNAPTP6 DNA polymerase-transactivated protein 6

Administration/Pharmaceutical Compositions

The invention provides for pharmaceutical compositions comprising theanti-IFN-α/ω antibody of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, and a pharmaceutically acceptable carrier. For therapeutic use,anti-IFN-α/ω antibody of the invention may be prepared as pharmaceuticalcompositions containing an effective amount of anti-IFN-α/ω antibody asan active ingredient in a pharmaceutically acceptable carrier. The term“carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the active compound is administered. Such vehicles may be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. For example, 0.4% saline and 0.3% glycine canbe used. These solutions are sterile and generally free of particulatematter. They may be sterilized by conventional, well-known sterilizationtechniques (e.g., filtration). The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the molecules or antibodies of the invention insuch pharmaceutical formulation may vary widely, i.e., from less thanabout 0.5%, usually to at least about 1% to as much as 15 or 20%, 25%,30%, 35%, 40%, 45% or 50% by weight and will be selected primarily basedon required dose, fluid volumes, viscosities, etc., according to theparticular mode of administration selected. Suitable vehicles andformulations, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in e.g. Remington: The Science andPractice of Pharmacy, 21^(st) Edition, Troy, D. B. ed., LipincottWilliams and Wilkins, Philadelphia, Pa. 2006, Part 5, PharmaceuticalManufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration of the anti-IFN-α/ω antibody in the methodsof the invention described herein, and in some embodiments of each andevery one of the numbered embodiments listed below, may be any suitableroute such as parenteral administration, e.g., intradermal,intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary,transmucosal (oral, intranasal, intravaginal, rectal) or other meansappreciated by the skilled artisan, as well known in the art.

The anti-IFN-α/ω antibody in the methods of the invention describedherein, and in some embodiments of each and every one of the numberedembodiments listed below, may be administered to a patient by anysuitable route, for example parentally by intravenous (i.v.) infusion orbolus injection, intramuscularly or subcutaneously or intraperitoneally.i.v. infusion may be given over for, example, 15, 30, 60, 90, 120, 180,or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

The dose given to a patient having an immune-mediated inflammatorydisease or an autoimmune disease such as lupus is sufficient toalleviate or at least partially arrest the disease being treated(“therapeutically effective amount”) and may be sometimes 0.005 mg/kg toabout 100 mg/kg, e.g. about 0.05 mg/kg to about 20 mg/kg or about 0.1mg/kg to about 20 mg/kg, or about 1 mg to about 20 mg/kg, or about 4mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or, e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for exampleabout 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70,80, 90 or 100 mg/kg.

A fixed unit dose may also be given, for example, 50, 100, 200, 500 or1000 mg, or the dose may be based on the patient's surface area, e.g.,500, 400, 300, 250, 200, or 100 mg/m². Usually between 1 and 8 doses,(e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat theimmune-mediated inflammatory disease, such as lupus, but 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.

The administration of the anti-IFN-α/ω antibody in the methods of theinvention and in some embodiments of each and every one of the numberedembodiments listed below, may be repeated after one day, two days, threedays, four days, five days, six days, one week, two weeks, three weeks,one month, five weeks, six weeks, seven weeks, two months, three months,four months, five months, six months or longer. Repeated courses oftreatment are also possible, as is chronic administration. The repeatedadministration may be at the same dose or at a different dose. Forexample, the anti-IFN-α/ω antibody in the methods of the invention maybe administered at 0.1 mg/kg, at 1 mg/kg, at 5 mg/kg, at 8 mg/kg or at16 mg/kg at weekly interval for 8 weeks, followed by administration at 8mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks,followed by administration at 8 mg/kg or at 16 mg/kg every four weeks byintravenous infusion.

The anti-IFN-α/ω antibody may be administered in the methods of theinvention and in some embodiments of each and every one of the numberedembodiments listed below, by maintenance therapy, such as, e.g., once aweek for a period of 6 months or more.

For example, the anti-IFN-α/ω antibody in the methods of the inventionand in some embodiments of each and every one of the numberedembodiments listed below, may be provided as a daily dosage in an amountof about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, onat least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20after initiation of treatment, or any combination thereof, using singleor divided doses of every 24, 12, 8, 6, 4, or 2 hours, or anycombination thereof.

The anti-IFN-α/ω antibody in the methods of the invention and in someembodiments of each and every one of the numbered embodiments listedbelow, may also be administered prophylactically in order to reduce therisk of developing the immune-mediated inflammatory disease or anautoimmune disease such as lupus, delay the onset of the immune-mediatedinflammatory disease of the autoimmune disease, and/or reduce the riskof recurrence when the immune-mediated inflammatory disease or theautoimmune disease such as lupus is in remission.

Thus, a pharmaceutical composition of the invention for intramuscularinjection may be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg/kg, e.g. about 50 ng to about 30mg/kg or more preferably, about 5 mg to about 25 mg/kg, of theanti-IFN-α/ω antibody of the invention.

For example, a pharmaceutical composition comprising the anti-IFN-α/ωantibody in the methods of the invention described herein, and in someembodiments of each and every one of the numbered embodiments listedbelow, for intravenous infusion may be made up to contain about 200 mlof sterile Ringer's solution, and about 8 mg to about 2400 mg, about 400mg to about 1600 mg, or about 400 mg to about 800 mg of theanti-INF-α/co antibody for administration to a 80 kg patient. Methodsfor preparing parenterally administrable compositions are well known andare described in more detail in, for example, “Remington'sPharmaceutical Science”, 15th ed., Mack Publishing Company, Easton, Pa.

The “therapeutically effective amount” of the IFN-α/ω antibodies of theinvention effective in the treatment of an immune-mediated inflammatorydisease or an autoimmune disease may be determined by standard researchtechniques. For example, in vitro assays may be employed to helpidentify optimal dosage ranges. Optionally, the dosage of the IFN-α/ωantibodies of the invention that may be effective in the treatment ofimmune-mediated inflammatory diseases or autoimmune diseases such aslupus including SLE may be determined by administering the IFN-α/ωantibodies to relevant animal models well known in the art. Selection ofa particular effective dose can be determined (e.g., via clinicaltrials) by those skilled in the art based upon the consideration ofseveral factors. Such factors include the disease to be treated orprevented, the symptoms involved, the patient's body mass, the patient'simmune status and other factors known by the skilled artisan. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the severity of disease, and should bedecided according to the judgment of the practitioner and each patient'scircumstances. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. Theantibodies of the invention may be tested for their efficacy andeffective dosage using any of the models described herein.

The anti-IFN-α/ω antibody in the methods of the invention describedherein, and in some embodiments of each and every one of the numberedembodiments listed below, may be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional protein preparations andwell known lyophilization and reconstitution techniques can be employed.

The anti-IFN-α/ω antibody in the methods of the invention describedherein, and in some embodiments of each and every one of the numberedembodiments listed below, may be administered in combination with asecond therapeutic agent simultaneously, sequentially or separately.

The second therapeutic agent may be a corticosteroid, an antimalarialdrug, an immunosuppressant, a cytotoxic drug, or a B-cell modulator.

In some embodiments, the second therapeutic agent is prednisone,prednisolone, methylprednisolone, deflazcort, hydroxychloroquine,azathioprine, methotrexate, cyclophosphamide, mycophenolate mofetil(MMF), mycophenolate sodium, cyclosporine, leflunomide, tacrolimus,Rituximab™, or Belimumab™.

Further Embodiments of the Invention

Set out below are certain further embodiments of the invention accordingto the disclosures elsewhere herein. Features from embodiments of theinvention set out above described as relating to the invention disclosedherein also relate to each and every one of these further numberedembodiments.

-   1) An isolated monoclonal antibody that binds to and neutralizes a    biological activity of a human interferon omega (IFN-ω) and at least    three, four, five, six, seven, eight, nine, ten or eleven human    interferon alpha (IFN-α) subtypes.-   2) The antibody according to embodiment 1, wherein the biological    activity of the human IFN-ω and the human IFN-α subtypes is the    human IFN-ω or the human IFN-α subtype-induced expression of    secreted embryonic alkaline phosphatase (SEAP) under interferon    inducible ISG54 promoter in HEK293 cells stably expressing signal    transducer and activator of transcription 2 (STAT2), interferon    regulatory factor 9 (IRF9) and SEAP.-   3) The antibody according to embodiment 1 or 2, wherein the antibody    neutralizes the biological activity of the human IFN-ω with an IC₅₀    of at least about 1×10⁻⁹M or less, about 1×10⁻¹⁰ M or less, about    5×10⁻¹¹M or less, or about 1×10⁻¹¹M or less.-   4) The antibody according to any one of embodiments 1-3, wherein the    antibody neutralizes the biological activity of the human IFN-ω with    an IC₅₀ value of at least about 1×10⁻¹⁰ M or less.-   5) The antibody according to any one of embodiments 1-4, wherein the    antibody neutralizes the activity of the human IFN-ω with an IC₅₀    value of between about 1×10⁻¹⁰ M to about 6×10⁻¹²M.-   6) The antibody according to any one of embodiments 1-5, wherein the    antibody neutralizes the activity of at least three, four, five,    six, seven, eight, nine, ten or eleven human IFN-α subtypes with an    IC₅₀ value of at least about 1×10⁻¹⁰ M or less.-   7) The antibody according to embodiment 6, wherein the IFN-α    subtypes are selected from the group consisting of IFN-αA, IFN-αB2,    IFN-αC, IFN-αF, IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK, IFN-αWA    and IFN-α4a.-   8) The antibody according to embodiment 7, wherein the antibody    comprises heavy chain complementarity determining region (HCDR) 1    (HCDR1), 2 (HCDR2) and 3 (HCDR3) amino acid sequences of SEQ ID NOs:    109, 114 and 121, respectfully, and light chain complementarity    determining region (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) amino    acid sequences of SEQ ID NOs: 118, 119 and 120.-   9) The antibody according to any one of embodiments 1-5, wherein the    antibody neutralizes at least six human IFN-α subtypes selected from    the group consisting of IFN-αA, IFN-αB2, IFN-αC, IFN-αF, IFN-αG,    IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK, IFN-αWA and IFN-α4a.-   10) The antibody according to embodiment 9, wherein the antibody    comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and    the LCDR3 amino acid sequences of SEQ ID NOs: 109, 114, 121, 159,    119 and 160, respectively.-   11) The antibody according to any one of embodiments 1-5, wherein    the antibody neutralizes at least ten human IFN-α subtypes selected    from the group consisting of IFN-αA, IFN-αB2, IFN-αC, IFN-αF,    IFN-αG, IFN-αH2, IFN-αI, IFN-αJ1, IFN-αK, IFN-αWA and IFN-α4a.-   12) The antibody according to embodiment 11, wherein the antibody    binds human IFN-ω) of SEQ ID NO: 1 at least at amino acid residues    F27, L30 and R33.-   13) The antibody according to any one of embodiments 1-5, wherein    the antibody comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1,    the LCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 109,    114, 121, 161, 119 and 162, respectively.-   14) The antibody according to any one of embodiments 11-13, wherein    the antibody neutralizes at least the human IFN-α subtypes IFN-αA,    IFN-αB2, IFN-αC, IFN-αG, IFN-αH2, IFN-αJ1 and IFN-α4a.-   15) The antibody according to embodiment 14, wherein the antibody    further neutralizes IFN-αI, IFN-αK or IFN-αWA.-   16) The antibody according to any one of embodiments 1-15, wherein    the antibody    -   a) inhibits leukocyte interferon-induced IP-10 release in whole        blood induced by 250 U/ml of interferon by about 50% or more in        the presence of 10 μg/ml antibody; or    -   b) inhibits systemic lupus erythematosus (SLE) immune        complex-induced IP-10 release in whole blood by about 50% or        more in the presence of 10 μ/ml antibody.-   17) The antibody according to any one of embodiments 1-16, wherein    the antibody comprises a heavy chain variable region (VH) amino acid    sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%    identical to SEQ ID NO: 28 and a light chain variable region (VL)    amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,    97%, 98% or 99% identical to SEQ ID NO: 150.-   18) The antibody according to any one of embodiments 1-17,    comprising    -   a) the HCDR1 amino acid sequences of SEQ ID NOs: 109;    -   b) the HCDR2 amino acid sequences of SEQ ID NOs: 111, 112 or        113;    -   c) the HCDR3 amino acid sequences of SEQ ID NOs: 115 or 116;    -   d) the LCDR1 amino acid sequences of SEQ ID NOs: 76, 77, 78, 79,        80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91;    -   e) the LCDR2 amino acid sequences of SEQ ID NOs: 93, 94 or 95;        and    -   f) the LCDR3 amino acid sequences of SEQ ID NOs: 96, 97, 98, 99,        100, 101, 102, 103, 104, 105, 106 or 107.-   19) The antibody according to embodiment 18, comprising the HCDR1,    the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 sequences    of SEQ ID NOs:    -   a) 109, 113, 116, 77, 93 and 104, respectively;    -   b) 109, 113, 116, 85, 93 and 96, respectively;    -   c) 109, 113, 115, 79, 95 and 107, respectively;    -   d) 109, 113, 116, 76, 93 and 103, respectively;    -   e) 109, 113, 115, 85, 93 and 96, respectively;    -   f) 109, 113, 115, 89, 95 and 100, respectively;    -   g) 109, 113, 116, 86, 93 and 105, respectively;    -   h) 109, 113, 115, 76, 93 and 103, respectively;    -   i) 109, 113, 116, 80, 93 and 97, respectively;    -   j) 109, 113, 116, 84, 93 and 97, respectively;    -   k) 109, 113, 116, 90, 93 and 97, respectively;    -   l) 109, 113, 116, 88, 93 and 102, respectively;    -   m) 109, 113, 116, 87, 93 and 105, respectively;    -   n) 109, 113, 116, 91, 93 and 106, respectively;    -   o) 109, 113, 115, 80, 93 and 97, respectively;    -   p) 109, 113, 116, 83, 93 and 101, respectively;    -   q) 109, 113, 116, 82, 94 and 98, respectively;    -   r) 109, 113, 115, 78, 95 and 100, respectively;    -   s) 109, 111, 116, 81, 93 and 106, respectively;    -   t) 109, 113, 116, 82, 94 and 99, respectively;    -   u) 109, 113, 115, 81, 93 and 106, respectively;    -   v) 109, 112, 116, 81, 93 and 106, respectively; or    -   w) 109, 113, 116, 81, 93 and 106, respectively.-   20) The antibody according to any one of embodiments 1-19, wherein    the antibody is humanized or human.-   21) The antibody according to embodiment 20, wherein the human    antibody heavy chain variable region framework is derived from human    germline gene IGHV5-51 (SEQ ID NO: 155).-   22) The antibody according to embodiment 21, wherein the human    antibody light chain variable region framework is derived from human    germline gene IGKV1D-39 (SEQ ID NO: 156).-   23) The antibody according to any one of embodiments 1-22, wherein    the antibody is of IgG1, IgG2, IgG3 or IgG4 subtype.-   24) The antibody according to embodiment 23, wherein the antibody    has at least one substitution in an Fc region.-   25) The antibody according to embodiment 24, wherein the wherein the    substitution comprises a substitution M252Y/S254T/T256E,    V234A/G237A/P238S/H28AN309L/A330S/P331S or P238S/L234A/L235A,    wherein residue numbering is according to the EU numbering.-   26) The antibody according to any one of embodiments 1-26,    comprising a heavy chain variable region (VH) and a light chain    variable region (VL), wherein the a) VH comprises the amino acid    sequence of SEQ ID NOs: 28, 31, 157 or 158.-   27) The antibody according to embodiment 26, wherein the VL    comprises the amino acid sequence of SEQ ID NOs: 35, 39, 40, 42, 46,    52, 53, 54, 57, 61, 62, 68, 71, 73, 75, 135 or 150.-   28) The antibody according to embodiment 27 comprising the VH and    the VL of SEQ ID NOs:    -   a) 28 and 40, respectively;    -   b) 28 and 39, respectively;    -   c) 31 and 62, respectively;    -   d) 28 and 54, respectively;    -   e) 31 and 39, respectively;    -   f) 31 and 68, respectively;    -   g) 28 and 42, respectively;    -   h) 31 and 54, respectively;    -   i) 28 and 53, respectively;    -   j) 28 and 73, respectively;    -   k) 28 and 75, respectively;    -   l) 28 and 52, respectively;    -   m) 28 and 35, respectively;    -   n) 28 and 135, respectively;    -   o) 31 and 53, respectively;    -   p) 28 and 46, respectively;    -   q) 28 and 61, respectively;    -   r) 31 and 57, respectively;    -   s) 157 and 71, respectively;    -   t) 28 and 150, respectively;    -   u) 31 and 71, respectively;    -   v) 158 and 71, respectively; or    -   w) 28 and 71, respectively.-   29) The antibody according to any one of embodiments 1-28, wherein    the antibody is bispecific.-   30) The antibody according to embodiment 29, wherein the antibody    binds BLyS, CD40L, IL-6, CD27, BDCA2, IL-12, IL-23, IFN-αD, IL-17,    CD20, IL-10, CD22, IL-21, ICOS, ICOSL or IFN-γ.-   31) A pharmaceutical composition comprising the antibody according    to any one of embodiments 1-30 and a pharmaceutically accepted    carrier.-   32) A polynucleotide encoding the antibody VH or VL or the antibody    VH and VL of any one of embodiments 1-28.-   33) A vector comprising the polynucleotide of embodiment 32.-   34) A host cell comprising the vector of embodiment 33.-   35) A method of producing the antibody of embodiment 19, comprising    culturing the host cell of embodiment 33 in conditions that the    antibody is expressed, and recovering the antibody produced by the    host cell.-   36) The antibody according to any one of embodiments 1-30 for use in    the treatment of an immune-mediated inflammatory disease or an    autoimmune disease.-   37) The antibody according to embodiment 36 for use of    -   a) the immune-mediated inflammatory disease or the autoimmune        disease, wherein the immune-mediated inflammatory disease or the        autoimmune disease is optinally lupus, psoriasis, immune        thrombocytopenia (ITP), Aicardi-Goutieres syndrome (AGS),        systemic sclerosis, Sjögren's syndrome, myositis, common        variable immune deficiency (CVID), autoimmune thyroid disease,        type I diabetes, rheumatoid arthritis, transplant rejection or        graft versus host disease (GVHD);    -   b) chronic viral infection, wherein the chronic viral infection        is optionally HIV or hepatitis C infection.-   38) The antibody according to any one of embodiments 1-30 for use in    the treatment of lupus.-   39) The antibody according to embodiment 38 for use of lupus,    wherein lupus is systemic lupus erythematosus (SLE) or cutaneous    lupus erythematosus (CLE).-   40) The antibody according to any one of embodiments 1-30 for use in    the treatment of an immune-mediated inflammatory disease or lupus,    wherein the patient to be treated has    -   a) lupus nephritis; or    -   b) exhibits a Type I interferon signature.-   41) The antibody according to any one of embodiments 1-30 for use    according to embodiments 37-40 in combination with a second    therapeutic agent.-   42) The antibody according to embodiment 41, wherein the second    therapeutic agent is    -   a) an antibody that binds BLyS, CD40L, IL-6, CD27, BDCA2, IL-12,        IL-23, IFN-αD, IL-17, CD20, IL-10, CD22, IL-21, ICOS, ICOSL or        IFN-γ;    -   b) a corticosteroid, an antimalarial drug, an immunosuppressant,        a cytotoxic drug, or a B-cell modulator; or    -   c) prednisone, prednisolone, methylprednisolone, deflazcort,        hydroxychloroquine, azathioprine, methotrexate,        cyclophosphamide, mycophenolate mofetil (MMF), mycophenolate        sodium, cyclosporine, leflunomide, tacrolimus, Rituximab™ or        Belimumab™-   43) The antibody according to any one of embodiments 1-30, wherein    the antibody does not neutralize IFN-αD, IFN-α1 and/or IFN-β.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

Materials and Methods

ISRE Reporter Gene Assay (“ISRE Reporter Gene Assay”)

HEK-Blue™ IFN-α/β cells (InvivoGen, San Diego, Calif.) engineered toexpress a fully active type I IFN signaling pathway (stably expressingSTAT2 and IRF9) and transfected with a SEAP reporter gene under thecontrol of the IFN-α/β inducible ISG54 promoter was used. The cells weregrown in collagen type I coated T150 flasks in Dulbecco's modified eaglemedia with 10% fetal bovine serum, 100 ug/ml blasticidin and 30 ug/mlzeocin at 37° C., 5% CO₂. Cells were harvested and plated in 384-wellplates at 50 μl per well at 50,000 cells per ml. Plated cells wereincubated at 37° C., 5% CO₂ for 24 hr. Tested interferon samples wereprepared and diluted in spent HEK ISRE serum free medium, and 50 μl ofIFN sample was added to each well. Plated cells were incubated at 37°C., 5% CO₂ for 20 hr. Alkaline phosphatase was detected from 20 μl ofplated cell supernatants with 60 μl/well QUANTI-Blue™ resuspended infiltered water after incubation for 20 min at room temperature. Opticaldensity was read on a Biotek Synergy plate reader at 650 nm.

Some ISRE reporter gene assays were done in 96-well plates as follows:HEK-Blue™ IFN-α/β cells (InvivoGen, San Diego, Calif.) were plated at50,000 cells per well in 100 μl of selection free media(DMEM+Glutamax/10% FBS, Gibco) and allowed to incubate overnight at 37°C. The next day, type I IFN stimuli were prepared (i.e. recombinantinterferon, leukocyte IFN, IC induced IFN preps, serum, etc) with orwithout type I IFN inhibitors in a separate 96 well U-bottom transferplate (BD Falcon) and prewarmed at 37° C. for 10 minutes. A plate ofcells was removed from incubator and media was removed and replaced with100 μl of appropriate treatments prepared in 96 well U-bottom transferplate. Cells were placed back at 37° C. for 24 hours. The next day, 40μl of supernatant was transferred to a 96 well flat bottom plate (BDFalcon) containing 160 μl of QUANTI-Blue™ SEAP substrate (Invivogen).Plate was allowed to develop for about 15 minutes at which time it wasread using a spectrometer at an absorbency of 650 nm.

Example 1. Soluble IFN-ω is Present and Active in the Blood of SLEPatients

Plasma from two independent SLE cohorts from Nanjing China and serumcollected from a Caucasian cohort in the USA were analyzed for solubleIFN-ω and IFN-α using a multiplex ELISA using a VeriPlex humaninterferon multiplex ELISA kit (PBL Assay Science, cat no 51500-1)according to manufacturer's instructions. The multiplex ELISA detectsmany, but not all of the IFN-α subtypes and may not accurately reflectquantitative differences between total IFN-α levels versus IFN-ω.

IFN-ω, in addition of IFN-α, was found to be elevated in certainpatients from both Nanjing China cohort (FIG. 1A) and Caucasian cohort(FIG. 1B) from each cohort. FIG. 1A shows results from only thosepatients that were found to have elevated IFN-α or IFN-ω. Serum samplesfrom the Caucasian group were further screened for IFN-I activity usingan ISRE reporter gene assay. Donors exhibiting the greatest amount ofdetectable IFN protein by ELISA also demonstrated the greatest level ofISRE induction in the reporter gene assay (FIG. 1C).

Example 2. Combined Blockade of IFN-ω and IFN-α Results in GreaterInhibition of SLE Immune Complex-Induced IFN than IFN-α Blockade Alone

Effect of inhibition of IFN-α alone or both IFN-ω and IFN-α to reduceSLE immune complex-induced IFN, a stimulus better representing the typeI IFN milieu present in SLE, was evaluated. SLE immune complex-inducedIFN was prepared by stimulating human PBMCs with immune complexesprepared from two individual SLE donors and this conditioned media wasutilized in a type I IFN-inducible reporter gene assay (ISRE reportergene assay) in the presence of IFN inhibitors and controls.

Immune Complex Preparation

SLE donor 232 and 293 plasma (prescreened for IFN activity) and healthycontrol plasma (Astarte Biologics) was utilized for IgG purificationusing protein A/G columns (Thermo Scientific, Cat#89958) according tothe manufacturer's instructions. Serum from a pooled healthy donorpreparation (Life Technologies, Cat#34005100) was used for purificationof healthy control IgG. To create lysates for immune complex formation,HEK293T cells (ATCC, Cat# CRL-3216) were concentrated to 5×10⁷ cells/mlin 1×DPBS (Life Technologies, Cat#14190-250). To create lysates, freezethawing was performed for 4 cycles of 10 minutes, freezing at −80° C.and thawing at 37° C., except for an initial freezing of 30 min. After4^(th) freeze-thaw, cell debris was removed by centrifugation at 400×gfor 5 minutes. Purified IgG preparations and cell lysates were thenquantitated using a BCA protein assay (Pierce, Cat#23225) according tomanufacturer's instructions. To create immune complexed stimulatedconditioned media preparations, PBMCs from healthy donor sodiumheparinized blood were isolated using Cell Preparation tubes (BDVacutainer, Cat#362753), resuspended in RPMI 1640 (Life Technologies,Cat#11875-085)+10% FBS (Life Technologies, Cat#16140-063) media at 2×10⁶cells/ml and plated in 6 well plates in a volume of 2 ml/well. PurifiedIgG from SLE and healthy serum was premixed with cell lysates atequivalent concentrations of 500 ug/ml each and incubated at RT for 30minutes and then added to PBMCs in a volume of 2 ml per well andincubated for 24 hours at 37° C. Plates were centrifuged at 1000 rpm for5 minutes and PBMC immune complex-stimulated conditioned media wascollected, aliquoted, and stored at −80° C. for future use.

Activity Assay

HEK-Blue IFNα/β cells (Invivogen) were plated in a 96 well flat bottomplate at 50,000 cells per well in 200 μl DMEM (Life Technologies)+10%fetal bovine serum (Life Technologies) and incubated for 5 hours at 37°C. to allow cells to adhere to plate. After 5 hours, Hek-Blue cells wereremoved from incubator and supernatants were replaced with a 1:6dilution of donor 232 PBMC conditioned media or a 1:81 dilution of donor293 conditioned media (using HEK-Blue cell culture media as a diluent)with or without the following treatments: broad anti-IFN-α antagonistmAb (M24, human IgG1) at 0.4, 2, 10, 50, and 100 μg/ml along with afixed concentrations of 20 μg/ml isotype control (R&D Systems, murineIgG1), 100 μg/ml anti-IFN-α combined with 20 μg/ml anti-IFN-ω antagonistmAb (eBioscience, clone OMG5, murine IgG1), or 100 μg/ml human IgG1isotype control (Southern Biotech) combined with 20 μg/ml murine IgG1isotype control. Cells were incubated overnight at 37° C. The next day,40 μl of cell supernatant from each well was removed and added to 160 μlof Quanti-Blue alkaline phosphatase substrate (Invivogen) in a separate96 well flat bottom plate. Supernatants were allowed to react with thesubstrate for 10 minutes at which time the plate was read on aspectrophotometer at 650 nm wavelength. Optical densities were plottedin GraphPad Prism

The additional blockade of IFN-ω in the presence of IFN-α antagonistresulted in enhanced suppression of SLE-relevant IFN-I activity thanblockade of IFN-α alone (FIG. 2). As expected, conditioned media fromPBMCs stimulated with immune complexes from healthy donor (HV ICConditioned media) did not have detectable ISRE activity indicating theinterferogenic potential of SLE patient immune complexes.

Example 3. Immunomodulatory Effects of IFN-ω are Similar to Those ofIFN-α

Ability of IFN-ω to induce chemokine secretion, IFN gene signature,dendritic cell maturation and activation, and B-cell maturation wasevaluated in comparison to IFN-α. In these studies, IFN-αA and IFN-α2,two of the most widely used therapeutic IFN-α molecules, were primarilyused as representative IFN-α subtype controls. In some assays, IFN-αB2was used.

Induction of Chemokine Secretion and IFN Gene Signature

PBMCs isolated from 6 individual healthy human donors were stimulatedwith IFN-αA (IFN-α2) or IFN-ω, and the supernatants and pellets werecollected for analyses. 3, 6 and 24 hours post-treatment. A panel of 25cytokines were measured from the supernatants using Luminex immunoassay:IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12,IL-13, IL-15, IL-17, TNFα, IFN-α, IFN-γ, GM-CSF, MIP-1a, MIP-1β, IP-10,MIG, Eotaxin, RANTES, and MCP-1. IFN-ω) and IFN-α2 both enhanced thelevel of detectable IP-10, MCP-1, IL-1RA, IL-6, MIP-1α, and MIP-1β. FIG.3 shows the induction of IP-10 by IFN-ω) and IFN-α2. IL-8 secretion wasreduced by both treatments in these experiments. IL-2R, IL-12 and RANTESlevels were not altered by IFN-α or IFN-ω) treatment (with the exceptionof one donor which had an increase in RANTES only). All other analytesin the cytokine panel did not change with respect to IFN-α or IFN-ω)treatment or were below the limit of detection.

Collected pellets were processed for RNA and evaluated using a 21-geneIFN panel signature by microarray to evaluate possible similaritiesand/or differences in IFN-ω) and IFN-α induced expression. Human PBMCstreated with IFN-ω) exhibited neary indistinguishable qualitative andkinetic gene expression responses as compared to IFN-αA-treated cells.92.5% of genes modulated by IFN-αA treatment versus untreated controlwere also modulated by IFN-ω) treatment at 3 h. At the 6 and 24 hpost-treatment time points, 97.83% and 99.25% of genes modulated byIFN-α treatment were also modulated by IFN-ω, respectively (data notshown).

In summary, IFN-α and IFN-ω induced indistinguishable qualitativecytokine release and gene expression profiles between PBMC preparationsobtained from 6 individual healthy human donors suggesting that they mayconfer similar immunomodulatory effects.

IFN-ω Induces Differentiation of Dendritic Cells which is Inhibited byIFN-ω Blocking Antibodies

Ability of IFN-ω and IFN-α to induce monocyte to DC differentiation andthe functionality was evaluated.

Purified monocytes were differentiated to DC in the presence of GM-CSFalone or with IFN-α or IFN-ω in the presence or absence of 50 μg/mlanti-IFN-α or anti-IFN-ω for 3 days using standard methods. Cells wereharvested and analyzed for surface marker expression by 8-color FACS.Both IFN-α and IFN-ω) induced characteristic DC surface markerexpression CD83, and CD80, CD86, CD40, CD11c, and reduced expression ormonocyte marker CD14. Addition of either anti-IFN-α or anti-IFN-ω) atconcentration 50 μg/ml at the beginning of culture partially inhibitedDC differentiation while the isotype antibody had no effect (data notshown).

Mixed lymphocyte reaction (MLR) was used to demonstrate thefunctionality of the differentiated DCs. The differentiated DCs wereharvested, washed, resuspended in fresh media, and cultured withpurified CD4+ T cells at DC:CD4⁺ T cell ratios of 1:10, 1:20, and 1:100.On day 6 supernatants were collected and analyzed for secreted cytokinesusing a multiplex beads assay for 26 cytokines/chemokines. DCsdifferentiated in the presence of either IFN-α or IFN-ω) activated CD4⁺cells as shown by secretion of T cell specific cytokines IFN-γ andIL-17. DCs differentiated in the presence either the anti-IFN-α or theanti-IFN-ω) antibody did not induce CD4⁺ T cell activation. FIG. 4Ashows the lack of induced IFN-γ secretion from the CD4+ cells activatedby DCs differentiated in the presence of anti-IFN-α or anti-IFN-ωantibodies. FIG. 4B shows the lack of induced IL-17 secretion from theCD4+ cells activated by DCs differentiated in the presence of anti-IFN-αor anti-IFN-ω antibodies. IFN-α and IFN-ω also induced secretion ofIL-4, IL-5, IL-12p40 and IL-13 (data not shown). All culture conditionsincluded GM-CSF. Data is representative of 2 studies. Error barsindicate SD of Luminex triplicates. In the experiment shown in thefigure, data illustrated a DC to CD4 T cell ratio of 1:20 was used.

IFN-ω Induces T-Cell Independent B Cell Activation

B cells play a critically important role in lupus pathogenesis throughthe production of pathogenic autoantibodies and cytokines, and bypresenting antigens to T cells. B cell activation and functionalmaturation can occur in a T cell-dependent (TD) or T cell-independent(TI) fashion. In TI B cell responses, B cells are released fromT-dependent tolerance control as TLR ligands or dendritic cell-derivedcytokines are able to substitute for T cell help. In SLE, where both TLRligands (e.g. double-stranded DNA) and DC-derived cytokines (e.g. type IIFNs) are believed to contribute to disease pathogenesis, TI B cellactivation represents a likely relevant mechanism. Besides theproduction of autoantibodies, autoreactive B cells are thought to playimportant pathogenic roles by presenting autoantigens to T cells andsecreting pro-inflammatory cytokines. IFN-α has been reported to enhancethe production of pro-inflammatory IL-6 by human B cells activated withantibodies against the B cell receptor (BCR) and CpG (mimicking specificantigen and TLR-signals, respectively) in the absence of T cell-derivedfactors. Furthermore, co-culture with plasmacytoid DCs was shown toenhance B cell activation as determined by CD86 expression levels thatwas dependent on soluble factors. The ability of IFN-ω to enhanceCD86-expression and pro-inflammatory cytokine production by human Bcells was investigated using a T cell-independent culture systemPeripheral blood B cells were cultured with CpG (ODN-2006), anti-BCR,and CpG & anti-BCR, and varying concentrations of IFN-α2 (Alpha 2b) orIFN-ω as indicated (IFN concentrations in U/ml). CD86-expression (medianfluorescence levels) was determined after 3 days by flow cytometry, andsupernatants were analyzed by 26-plex Luminex immunoassay, includingIL-6. The results were expressed as mean values of duplicate samples±SD.

Dose-dependent IFN-ω-induced up-regulation of CD86 expression uponanti-BCR and anti-BCR/CpG stimulation was observed with both donorsamples tested, whereas co-culture of B lymphocytes without stimulusshowed only a weak effect. INF-ω induced CD86 expression to a similarextent than IFN-α2B. FIG. 5A shows the IFN-ω-induced CD86 expressionfrom B cells from one donor. IFN-ω also dose-dependently inducedIL-6-production upon CpG and anti-BCR/CpG stimulation to similar extentthan IFN-α2B with both donor samples tested. FIG. 5B shows theIFN-ω-induced IL-6 secretion from B cells from one donor.

IFN-ω Induces BLyS Secretion

BLyS (BAFF) is a B cell survival factor and a clinically validatedtarget in human SLE. IFN-α treatment has been found to induce BLyS geneexpression in vivo as determined by microarray and qPCR analysis ofPBMCs isolated from patients 24 h after dosing. Ability of IFN-ω toinduce secretion of BLyS was therefore assessed.

PBMCs were isolated from two different normal healthy donors. Equivalentconcentrations of IFN-ω and IFN-α were used to stimulate cells for 72hours at which time supernatants were collected and analyzed by ELISAfor soluble BLyS. Results were expressed as mean values of duplicatesamples±SD.

IFN-ω and IFN-α were similarly competent in inducing the secretion ofBLyS in human PBMCs in vitro. Results from one donor are shown in FIG.6.

Example 4 Generation of Human Type I IFN Antigens Used for Immunization,Phage Panning, Antibody Characterization, and Crystallography Studies

20 individual recombinant human type I IFN alphas shown in Table 4 werecloned and expressed in HEK 293 cells using standard methods usingsignal sequences, such as SEQ ID NOs: 21-25. The proteins are humanunless otherwise stated. To improve expression level and solubility, asingle amino acid mutant at position 80 of human IFN-ω, IFN-ω T80E wasgenerated and expressed in HEK 293 cells. The T80E IFN-ω variant (SEQ IDNO: 2) had comparable activity to the wild type protein. IFN-αD andIFN-α1 differ by one amino acid at position 114 (valine vs alanine).Alpha A and Alpha 2 differ by one amino acid at position 23 (lysine inAlpha A vs. arginine in Alpha 2). Alpha 4 has two forms, 4a and 4b thatdiffer by two amino acids at position 51 (alanine in Alpha 4a andthreonine in Alpha 4b) and 114 (glutamate in Alpha 4a vs valine in Alpha4b). These variations are located outside the receptor binding regionand do not affect activity. Antibodies were found to neutralize thesepairs of variants (αD/α1, αA/α2 and α4a/α4b) equally well andsubsequently in some experiments only one antigen of each pair was used.

TABLE 4 GenBank Accession SEQ IFN Alternative Number ID Protein NameAdopted NO: IFN-αA IFN-α2a V00549 5 IFN-αB2 IFN-α8 X03125 6 IFN-αCIFN-α10 NM_002171.1 7 IFN-αD Val114 IFN-α1 V00538 8 IFN-αF IFN-α21V00540 9 IFN-αG IFN-α5 X02956 10 IFN-αH2 IFN-α14 X02959 11 IFN-αIIFN-α17 V00532 12 IFN-αJ1 IFN-α7 X02960 13 IFN-αK IFN-α6 X02958 14IFN-α4b IFN-α4 X02955 15 IFN-αWA IFN-α16 X02957 16 IFN-α2 IFN-α2bV00548, 17 NM_00605.2 IFN-α1 A1a114 IFN-αD J00210 18 IFN-α4a IFN-αM1NM_021068 19 IFN-β V00534 20 IFN-ω NM_002177.1 1 IFN-ω T80E 2 ChimpIFN-ω XM_528554.1 3 Cyno IFN-ω NA 4

Example 5. Generation of Antibodies Binding to IFN-α and IFN-ω

IFN-α and IFN-ω-binding Fabs were selected from de novo pIX phagedisplay libraries as described in Shi et al., J Mol Biol 397:385-96,2010; Int. Pat. Publ. No. WO2009/085462; U.S. Pat. Publ. No.US2010/0021477). Briefly, the libraries were generated by diversifyinghuman scaffolds where germline VH genes IGHV1-69*01, IGHV3-23*01, andIGHV5-51*01 were recombined with the human IGHJ-4 minigene via the H3loop, and human germline VLkappa genes O12 (IGKV1-39*01), L6(IGKV3-11*01), A27 (IGKV3-20*01), and B3 (IGKV4-1*01) were recombinedwith the IGKJ-1 minigene to assemble complete VH and VL domains. Thepositions in the heavy and light chain variable regions around H1, H2,L1, L2 and L3 loops corresponding to positions identified to befrequently in contact with protein and peptide antigens were chosen fordiversification. Sequence diversity at selected positions was limited toresidues occurring at each position in the IGHV or IGLV germline genefamilies of the respective IGHV or IGLV genes. Diversity at the H3 loopwas generated by utilizing short to mid-sized synthetic loops of lengths7-14 amino acids. The amino acid distribution at H3 was designed tomimic the observed variation of amino acids in human antibodies. Librarydesign is detailed in Shi et al., J Mol Biol 397:385-96, 2010. Thescaffolds utilized to generate libraries were named according to theirhuman VH and VL germline gene origin. The three heavy chain librarieswere combined with the four germline light chains or germline lightchain libraries to generate 12 unique VH:VL combinations for panningexperiments against IFN-α and IFN-ω.

The libraries were panned against either biotinylated human IFN-α2 orbiotinylated human IFN-αG. After three rounds of panning, a polyclonalphage ELISA using human IFN-α2, IFN-αG and cynomolgus IFN-ω) as antigenswas performed to detect the specific enrichment of individual panningexperiments. The phage collected from those panning experiments whichdemonstrated enrichment for binders to IFN-α2, IFN-αG and IFN-ω werefurther screened with a monoclonal Fab ELISA in which Fab proteinsexpressed from individual Fab clones were used as binders. The Fabclones with binding signal to 20 nM biotinylated antigen three timeshigher than the negative control were selected for secondary Fabscreening. Select Fabs were cloned into IgG1/κ background andcharacterized further using ProteOn and ISRE reporter gene assay. Fromthese assays, mAb IFWM371 was selected for further engineering andaffinity maturation.

Table 5 shows affinities (K_(D)) and IC₅₀ values for IFWM371 as measuredusing ProteOn and ISRE reporter gene assay for various Type I IFNs aswell as IFN-β. Except IFN-α1 (IFN-αD), IFWM371 bound to all humanIFN-alpha proteins tested ranging from 179 pM-10 nM. The antibodies didnot bind IFN-α1 (IFN-αD). The antibody bound also human, chimpanzee andcynomolgus IFN-ω but did not bind IFN-β. IFWM371 demonstratedneutralizing activity to all tested IFN-α molecules except IFN-α1 (αD),which the antibody did not neutralize. IFWM371 contains the VH IFWH591(SEQ ID NO: 28) and the VL PH9L4 (germline O12) (SEQ ID NO: 29.

TABLE 5 K_(D) IC₅₀ (pM) (nM) IFN-αA 813 8.4 IFN-αB2 1140 19.3 IFN-αC1670 53.9 IFN-αD NB NN IFN-αF 5310 16 IFN-αG 1110 12.9 IFN-αH2 179 9.6IFN-αJ1 10800 35.7 IFN-αK 245 7.3 IFN-αWA 3180 74.2 IFN-α4a 5390 32.8IFN-β NB NN chimp IFN-ω 1080 cyno IFN-ω 887 human IFN-ω ND 43.9 NB nobinding ND not done NN non-neutralizing

Example 6. Crystal Structure of IFWM371 in Complex with IFN-ω T80E

In order to reveal the epitope and paratope, the structural basis forits broad binding specificity to IFN-α subtypes and IFN-ω, and toprovide support for engineering to improve affinity and specificity, thecrystallography study of human IFN-ω T80E in complex with Fab of IFWM371was performed.

His-tagged Fab IFWM371 (IgG1/kappa isotype) was cloned and expressed inHEK293 cells and purified using affinity, ion exchange andsize-exclusion chromatography. The Fab was received in 20 mM Tris pH7.4, 50 mM NaCl. Human IFN-ω T80E variant (hereafter simply IFN-ω) witha C-terminal 6xHis-Tag was expressed in HEK293 cells. The protein wasreceived in 20 mM Tris, pH 7.4, 50 mM NaCL.

The complex was prepared by mixing of IFN-ω with Fab IFWM371 in molarratio of 1.2:1.0 (excess IFN-ω), incubated at 4° C. overnight, andpurified on Superdex 200 column equilibrated with 20 mm HEPES pH 7.5,0.25 M NaCl, then concentrated to 9.96 mg/ml using Amicon-Ultra 10 kDacutoff. Crystals suitable for X-diffraction were obtained from 20% PEG3K, 0.2M ammonium phosphate dibasic with MMS seeding (Obmolova, G.,Malia, T. J., Teplyakov, A., Sweet, R. & Gilliland, G. L. (2010).Promoting crystallization of antibody-antigen complexes via microseedmatrix screening. Acta Crystallogr D Biol Crystallogr 66, 927-33.).

For X-ray data collection, one crystal of IFN-ω/Fab IFWM371 complex wassoaked for a few seconds in the mother liquor (20% PEG 3350, 0.2 M(NH₄)₂HPO₄, pH 7.9) supplemented with 20% glycerol, and flash frozen inthe stream of nitrogen at 100 K. X-ray diffraction data were collectedusing a Rigaku MicroMax™-007HF microfocus X-ray generator equipped withan Osmic™ VariMax™ confocal optics, Saturn 944 CCD detector, and anX-Stream™ 2000 cryocooling system (Rigaku, Tex.). Diffractionintensities were detected over a 205° crystal rotation in quarter-degreeimages. The X-ray data were processed with the program XDS. X-ray datastatistics are given in Table 6.

The structure of the IFN-ω/Fab IFM371 complex was solved by molecularreplacement (MR) with Phaser. The search models for MR were the crystalstructure of Fab15 (PDB ID 3NA9; Luo, J., Obmolova, G., Huang, A.,Strake, B., Teplyakov, A., Malia, T., Muzammil, S., Zhao, Y., Gilliland,G. L. & Feng, Y. (2010). Coevolution of antibody stability and VkappaCDR-L3 canonical structure. J Mol Biol 402, 708-19) and IFN-α4A.However, an MR solution could not be obtained for IFN-ω due to severeinter-molecular clashes. Inspection of the electron density map phasedwith Fab IFWM371 alone showed the electron density for over half of theIFN-ω molecule is missing. However, the remaining part of the IFN-ωmolecule was readily fit in the density. The structure was then refinedwith PHENIX and model adjustments were carried out using COOT.

TABLE 6 Crystal data Space group C2 Unit cell dimensions a, b, c (Å)153.84, 69.84, 54.69 α, β, γ (°) 90, 106.87, 90 Asymmetric unit content1 complex X-ray data Resolution (Å) 50-1.81 (1.85-1.81)* Number ofmeasured reflections 175,220 (1,217) Number of unique reflections 43,466(588) Completeness (%) 85.20 (39.5) R_(merge) 0.056 (0.321) <I/σ> 16.1(3.1) B-factor (Wilson plot) (Å²) 20.1 Refinement Resolution (Å)30.6-1.81 (1.84-1.81) Number of refls used in refinement 43,463 (1113)Number of all atoms 4,594 Number of water molecules 481 Rcryst (%) 18.3(25.9) Rfree (%) 21.5 (38.1) RMSD bond lengths (Å) 0.002 RMSD bondangles (°) 0.73 RMSD B-factor main-chain (Å²) 5.6 Mean B-factor (Å²)26.0 Protein 23.2 Solvent 38.0 MolProbity [25] Clash score 6.8 Rotameroutliers (%) 1.2 Ramachandran favored (%) 98.5 Ramachandran outliers (%)0.0 Cβ deviation > 0.25 Å 0 *Values for high-resolution shell are inparentheses

The overall molecular structure of the IFN-ω/Fab IFWM371 complex isshown in FIG. 7A. There was one complex in the asymmetric unit. Themolecular model for the IFN-ω molecule included residues 23-39 and119-153, corresponding to helical segment AB and helices D and E.Residue numbering is according to IFN-ω amino acid sequence shown in SEQID NO: 1. The helices A, B and C and the connecting loops weredisordered. The Fab molecular model contained residues from 1 to 212 forthe light chain (SEQ ID NO: 29) and from 1 to 222 for the heavy chain(SEQ ID NO: 28). The C-terminal 6xHis tag, inter-chain disulfide bondand residues of 137-141 of the heavy chain were disordered. In addition,there were a number of water molecules at the antibody/antigen interfacethat formed an extensive H-bonding networks (FIG. 7B).

The observed parts of IFN-ω molecule were nearly identical to thecorresponding parts of full-length model of a published IFN-ω (PDB id3se4, Cα rmsd of 0.54 Å for 40 residues) and very similar to IFN-α2 withan average Cα rmsd of 0.42 Å (six IFN-α2 molecules, pdb code 1rh2) forabout 40 Cα atoms. The model for IFN-ω in the IFN-ω/Fab IFWM371contained only parts of helices C and D as well as connecting loop (loopAB). The other parts were absent in the electron density. Crystalpacking analyses showed that there was not enough room for the missinghelices. Careful analyses of the diffraction data indicated this was notan artifact due to abnormalities such as twinning or incorrect spacegroup assignment. Thus, it was most likely that the IFN-ω protein hadbeen cleaved during the crystallization process.

Fab IFWM371 recognized a conformational epitope that is composed ofresidues of the AB loop (between S25 and D35) and residues M146, andK150 of helix E (FIG. 8A). The paratope is composed of residues fromfive CDRs except LCDR2. The paratope residues form a series of pocketsinto which dock the side chains of residues F27, L30, and R33 of theshort AB helix of IFN-ω. FIG. 8B shows the paratope residues in VL andVH of IFWM371. The antibody and antigen interactions appear to be mostlyvan der Waals (vdw) and hydrophobic packing as well as H bonds betweenthe antibody and antigen. FIG. 8C shows a 2D Interaction map betweenIFN-ω and IFWM371 interactions. In the figure, IFN-ω epitope residuesare highlighted in grey, VL paratope residues are boxed, and VH paratoperesidues are circled. The figure demonstrates that most antigen/antibodyinteractions are formed by the three epitope residues F27, L30 and R33of the IFN-ω AB helix. Thus, this region of IFN-ω constitutes the mainpart of the epitope. Another feature of this complex is that watermolecules appeared to play a significant role mediating antigenrecognition. Three water clusters (WCs) were present at the interface.WC1 contributed to H bond interactions between HCDR3 and R34, F36 andE147 of IFN-ω. WC2 mediated VH/VL pairing and H bonding between Fv andthe main epitope residues L30, R33 and its neighbors. WC3 watermolecules were at the periphery of the interface, probably lessimportant for the interactions.

IFWM371 strongly binds a number of IFN-α subtypes and IFN-ω exceptIFN-αD or IFN-α1. IFWM371 does not bind IFN-β. The sequence alignment ofIFNs is shown in FIG. 9. The IFWM371 epitope residues are largelyconserved among the subtypes, suggesting that the broad specificity ofIFNM371 is a result of epitope conservation. IFN-αD or IFN-α1, however,to which IFWM371 does not bind to, contains S27 instead of F27, leadingto a loss of the majority of hydrophobic contacts of F27 side chain.Since F27 is docked in a deep pocket formed by residues of HCDR2, HCDR3and LCDR3, loss of the side chain contacts most likely accounts for thevery low or no binding by IFN-αD and IFN-α1 proteins. This also suggeststhat F27 is one of the binding “hot spot” residues. P26 is a residuethat is less well conserved. A His or Leu residue occupies this positionin several IFN-α subtypes. Because of the size and shape differences,this residue can significantly influence the local interactions betweenIFWM371 and IFN-α's with these mutations.

Example 7. Alanine Scan of IFWM371

Alanine scan of IFWM371 heavy and light chain CDR residues was conductedto guide subsequent affinity-maturation efforts. All residues in theCDRs of both heavy and light chains were replaced with alanine exceptsome low solvent exposure or non-solvent exposed residues. When nativeresidues at CDRs were alanine, they were replaced with Tyrosine and/orSerine and/or Aspartic acid. One position with possible developabilityliabilities (W104 in IFWH591, SEQ ID NO 28) was replaced with Alanine,Tyrosine, Serine, and Aspartic acid. The mutated mAbs were transientlyexpressed in HEK 293 cells and cell supernatants were tested for bindingactivity to a panel of IFNs by ELISA. Two V_(H) mutants, IFWH591 R59A(SEQ ID NO: 30) and IFWH591 N103A (SEQ ID NO: 31), had significantlyimproved binding compared to the parent mAb.

Example 8. Affinity-Maturation of IFWM371

Library Design

Two distinct V_(L) libraries (PH9L4L2 and PH9L4L3) were designed andused to affinity-mature IFWM371 light chain PH9L4 (O12) (SEQ ID NO: 29).The positions chosen for diversification of library PH9L4L2 were basedon residue positions frequently found in anti-protein and anti-peptidecomplexes. The residues used to diversify each position were encodedwithin the germline gene family of IGKV genes (Shi et α1 (2010) J. Mol.Biol. 397:385-96). The library complexity was limited to not exceed 10⁷library members so that the diversity could be fully assessed duringaffinity maturation (the actual library complexity: 3.5⁷). Table 7 showsthe library design diversification scheme for LCDR1 position 30, 31 and32, LCDR2 positions 50 and LCDR3 position 91, 92, 93, 94 and 96 of theV_(L) PH9L4 (O12) in the library. Residue numbering is according toKabat.

TABLE 7 Amino acid position on O12 (SEQ ID Diversified with NO: 29)amino acid Ser30 S, R, N, A, D Ser31 N, S, K, D, G Tyr32 Y, W, D, F, H,S, N, A, V Ala50 A, D, G, K, Y, F, T, N Ser91 Y, S, H, A Tyr92 Y, N, D,S, H, I, F, K, G, R, E Ser93 S, N, T, D, G, H, R Thr94 T, Y, L, V, F, S,R, G, P, I Leu96 W, Y, F, L, I, R, N

The residue positions to be diversified in the second light chainaffinity-maturation library, PH9L4L3, were chosen based on analysis ofstructures between antibody-protein complexes and the diversity in eachposition was designed based on analyzing antibody protein structures aswell as the amino acid usage in germline genes for each position (G.Raghunathan et al, Antigen-binding site anatomy and somatic mutations inantibodies that recognize different types of antigens. J. Mol Recognit.25:103-113 (2012). For LCDR3, diversity was extended beyond naturalrepertoire to ensure that each position has amino acids of differentbiochemical properties (i.e., polar/nonpolar, positively/negativelycharged). Additionally, the relative frequency of each amino acid perposition were varied which was made possible using the Sloning librarysynthesis technology. Table 8 shows the library composition of PH9L4L3.Residue numbering is according to Kabat.

TABLE 8 Amino acid position on O12 (SEQ ID NO: 29) Diversified withamino acid Ser30 S, R, N, A, D Ser31 N, S, T Tyr32 Y, N, D, S, R Tyr49Y, K, E, H Ala50 A, Y, W, S, G, N Ser91 S, H, W, Y, E, A, G, D, N, RTyr92 Y, S, H, W, E, A, G, D, N, R Ser93 S, H, W, Y, E, A, G, D, N, RThr94 T, S, H, W, Y, E, A, D, N, R, G Leu96 W, Y, F, L, I

Panning and Characterization

Affinity maturation libraries were generated by combining the lightchain libraries PH9L4L2 or PH9L4L3 with the parental heavy chain IFWH591(SEQ ID NO: 28). The libraries were then used for panning to select forhigh affinity antibodies. Some affinity-maturation panning experimentsresulted in biased improvements in binding either only to IFN-ω or onlyto a few IFN-α subtypes but not both. In order to generate broadlyneutralizing antibodies with improved IC₅₀ for most IFN-α subtypes andIFN-ω, a subset of IFN-α subtypes that were more diversified from eachother (IFN-α2, IFN-α4a, IFN-αF and IFN-αG) were panned alternativelywith cynomolgus monkey or human IFN-ω between each panning round. Atotal of three rounds of panning were carried out for each panningexperiment.

Fab proteins of individual clones were expressed in TG-1 E. coli andbacterial cell lysates were used for Fab ELISAs to determine theiraffinities to human IFN-α4a, IFN-αF and IFN-ω compared to IFWM371. SinceIFWM371 Fab bound these antigens weakly, Fab IFWF477 having higheraffinity to the antigens was used as the surrogate Fab for comparison.42 clones were identified that exhibited several folds higher bindingactivity than the surrogate Fab in ELISA. Some variants contained oneamino acid insertion on LCDR1 which was not part of the original librarydesign but was introduced during library synthesis. Overall, theaffinity maturation of the VL resulted in a significant improvement inbinding compared to the surrogate Fab. The best clones from the twolibraries showed over 23-fold higher binding activity to human IFN-ωthan the surrogate Fab IFWF477 respectively.

For further functional and biophysical characterization, total of 42light chains derived from the libraries were paired with the parentalheavy chain IFWH591 (SEQ ID NO: 28) as well as two V_(H) variants withimproved binding activity, IFWH624 (IFWH591 R59A, SEQ ID NO: 30) andIFWH629 (IFWH591 N103A, SEQ ID NO: 31), identified from the alaninescanning experiment described in Example 7. A total of 126 convertedmAbs (42 light chains paired with three heavy chains) were thenexpressed and characterized further. Table 9 shows the parental andselect affinity-matured antibodies and their heavy and light chainvariable regions.

TABLE 9 Antibody name Protein VL VH VH VL Protein amino acid PeptidePeptide SEQ ID SEQ ID cDNA name name name name NO: NO: IFWM371 IFWB351PH9L4 IFWH591 28 29 IFWM3301 IFWB3036 IFWL983 IFWH591 28 32 IFWM3302IFWB3037 IFWL991 IFWH591 28 33 IFWM3303 IFWB3038 IFWL992 IFWH591 28 34IFWM3304 IFWB3039 IFWL997 IFWH591 28 35 IFWM3305 IFWB3040 IFWL998IFWH591 28 36 IFWM3291 IFWB3026 IFWL999 IFWH591 28 37 IFWM3306 IFWB3041IFWL1000 IFWH591 28 38 IFWM3307 IFWB3042 IFWL1001 IFWH591 28 39 IFWM3308IFWB3043 IFWL1004 IFWH591 28 40 IFWM3309 IFWB3044 IFWL1006 IFWH591 28 41IFWM3310 IFWB3045 IFWL1007 IFWH591 28 42 IFWM3311 IFWB3046 IFWL1009IFWH591 28 43 IFWM3312 IFWB3047 IFWL1010 IFWH591 28 44 IFWM3313 IFWB3048IFWL1013 IFWH591 28 45 IFWM3314 IFWB3049 IFWL1014 IFWH591 28 46 IFWM3315IFWB3050 IFWL1017 IFWH591 28 47 IFWM3316 IFWB3051 IFWL1022 IFWH591 28 48IFWM3317 IFWB3052 IFWL1026 IFWH591 28 49 IFWM3318 IFWB3053 IFWL1038IFWH591 28 50 IFWM3319 IFWB3054 IFWL1041 IFWH591 28 51 IFWM3320 IFWB3055IFWL1047 IFWH591 28 52 IFWM3321 IFWB3056 IFWL1048 IFWH591 28 53 IFWM3322IFWB3057 IFWL1051 IFWH591 28 54 IFWM3323 IFWB3058 IFWL1053 IFWH591 28 55IFWM3325 IFWB3060 IFWL1060 IFWH591 28 56 IFWM3327 IFWB3062 IFWL1063IFWH591 28 57 IFWM3328 IFWB3063 IFWL1064 IFWH591 28 58 IFWM3329 IFWB3064IFWL1067 IFWH591 28 59 IFWM3330 IFWB3065 IFWL1071 IFWH591 28 60 IFWM3331IFWB3066 IFWL1073 IFWH591 28 61 IFWM3332 IFWB3067 IFWL1074 IFWH591 28 62IFWM3333 IFWB3068 IFWL1076 IFWH591 28 63 IFWM3334 IFWB3069 IFWL1082IFWH591 28 64 IFWM3335 IFWB3070 IFWL1084 IFWH591 28 65 IFWM3336 IFWB3071IFWL1085 IFWH591 28 66 IFWM3337 IFWB3072 IFWL1087 IFWH591 28 67 IFWM3338IFWB3073 IFWL1091 IFWH591 28 68 IFWM3339 IFWB3074 IFWL1093 IFWH591 28 69IFWM3340 IFWB3075 IFWL983 IFWH624 30 32 IFWM3341 IFWB3076 IFWL991IFWH624 30 33 IFWM3342 IFWB3077 IFWL992 IFWH624 30 34 IFWM3343 IFWB3078IFWL997 IFWH624 30 35 IFWM3344 IFWB3079 IFWL998 IFWH624 30 36 IFWM3292IFWB3027 IFWL999 IFWH624 30 37 IFWM3345 IFWB3080 IFWL1000 IFWH624 30 38IFWM3346 IFWB3081 IFWL1001 IFWH624 30 39 IFWM3347 IFWB3082 IFWL1004IFWH624 30 40 IFWM3348 IFWB3083 IFWL1006 IFWH624 30 41 IFWM3349 IFWB3084IFWL1007 IFWH624 30 42 IFWM3350 IFWB3085 IFWL1009 IFWH624 30 43 IFWM3351IFWB3086 IFWL1010 IFWH624 30 44 IFWM3352 IFWB3087 IFWL1013 IFWH624 30 45IFWM3353 IFWB3088 IFWL1014 IFWH624 30 46 IFWM3354 IFWB3089 IFWL1017IFWH624 30 47 IFWM3355 IFWB3090 IFWL1022 IFWH624 30 48 IFWM3356 IFWB3091IFWL1026 IFWH624 30 49 IFWM3357 IFWB3092 IFWL1038 IFWH624 30 50 IFWM3358IFWB3093 IFWL1041 IFWH624 30 51 IFWM3359 IFWB3094 IFWL1047 IFWH624 30 52IFWM3360 IFWB3095 IFWL1048 IFWH624 30 53 IFWM3361 IFWB3096 IFWL1051IFWH624 30 54 IFWM3364 IFWB3099 IFWL1060 IFWH624 30 56 IFWM3366 IFWB3101IFWL1063 IFWH624 30 57 IFWM3367 IFWB3102 IFWL1064 IFWH624 30 58 IFWM3368IFWB3103 IFWL1067 IFWH624 30 59 IFWM3369 IFWB3104 IFWL1071 IFWH624 30 60IFWM3370 IFWB3105 IFWL1073 IFWH624 30 61 IFWM3371 IFWB3106 IFWL1074IFWH624 30 62 IFWM3372 IFWB3107 IFWL1076 IFWH624 30 63 IFWM3374 IFWB3109IFWL1084 IFWH624 30 65 IFWM3375 IFWB3110 IFWL1085 IFWH624 30 66 IFWM3376IFWB3111 IFWL1087 IFWH624 30 67 IFWM3377 IFWB3112 IFWL1091 IFWH624 30 68IFWM3378 IFWB3113 IFWL1093 IFWH624 30 69 IFWM3379 IFWB3114 IFWL983IFWH629 31 32 IFWM3380 IFWB3115 IFWL991 IFWH629 31 33 IFWM3381 IFWB3116IFWL992 IFWH629 31 34 IFWM3382 IFWB3117 IFWL997 IFWH629 31 35 IFWM3383IFWB3118 IFWL998 IFWH629 31 36 IFWM3293 IFWB3028 IFWL999 IFWH629 31 37IFWM3384 IFWB3119 IFWL1000 IFWH629 31 38 IFWM3385 IFWB3120 IFWL1001IFWH629 31 39 IFWM3386 IFWB3121 IFWL1004 IFWH629 31 40 IFWM3387 IFWB3122IFWL1006 IFWH629 31 41 IFWM3388 IFWB3123 IFWL1007 IFWH629 31 42 IFWM3389IFWB3124 IFWL1009 IFWH629 31 43 IFWM3390 IFWB3125 IFWL1010 IFWH629 31 44IFWM3391 IFWB3126 IFWL1013 IFWH629 31 45 IFWM3392 IFWB3127 IFWL1014IFWH629 31 46 IFWM3393 IFWB3128 IFWL1017 IFWH629 31 47 IFWM3394 IFWB3129IFWL1022 IFWH629 31 48 IFWM3395 IFWB3130 IFWL1026 IFWH629 31 49 IFWM3396IFWB3131 IFWL1038 IFWH629 31 50 IFWM3397 IFWB3132 IFWL1041 IFWH629 31 51IFWM3398 IFWB3133 IFWL1047 IFWH629 31 52 IFWM3399 IFWB3134 IFWL1048IFWH629 31 53 IFWM3400 IFWB3135 IFWL1051 IFWH629 31 54 IFWM3401 IFWB3136IFWL1053 IFWH629 31 55 IFWM3403 IFWB3138 IFWL1060 IFWH629 31 56 IFWM3405IFWB3140 IFWL1063 IFWH629 31 57 IFWM3406 IFWB3141 IFWL1064 IFWH629 31 58IFWM3407 IFWB3142 IFWL1067 IFWH629 31 59 IFWM3408 IFWB3143 IFWL1071IFWH629 31 60 IFWM3409 IFWB3144 IFWL1073 IFWH629 31 61 IFWM3410 IFWB3145IFWL1074 IFWH629 31 62 IFWM3411 IFWB3146 IFWL1076 IFWH629 31 63 IFWM3413IFWB3148 IFWL1084 IFWH629 31 65 IFWM3414 IFWB3149 IFWL1085 IFWH629 31 66IFWM3415 IFWB3150 IFWL1087 IFWH629 31 67 IFWM3416 IFWB3151 IFWL1091IFWH629 31 68 IFWM3417 IFWB3152 IFWL1093 IFWH629 31 69 IFWM3418 IFWB3153IFWL1049 IFWH591 28 70 IFWM3419 IFWB3154 IFWL1049 IFWH629 31 70 IFWM3420IFWB3155 IFWL1049 IFWH624 30 70 IFWM3421 IFWB3156 IFWL984 IFWH591 28 71IFWM3423 IFWB3158 IFWL984 IFWH629 31 71

Affinities of the 126 generated mAbs to a panel of human IFN-ω and humanIFN-α subtypes were measured by ProteOn. The mAbs were transientlytransfected in triplicate along with controls in HEK 293E cells in48-well plates and cell supernatants were used in this experiment. Toincrease the assay throughput, only one concentration of the individualantigen was used. Table 10 shows the K_(D) values for the parentalIFWM371 and select affinity-matured antibodies. Most of the mAbs showedsignificant improvement of binding affinity to all antigens tested. Someof them showed more than 100-fold improvement over the parental mAb.

TABLE 10 K_(D) (pM) Protein cDNA IFN- IFN- IFN- IFN- IFN- IFN- IFN- IFN-IFN- name ω αC αF αJ1 α4a αB2 αG αWA α2 IFWM371 1060 865 775 4230 4180418 263 1390 116 IFWM3301 18 24 29 74 146 39 29 79 42 IFWM3302 52 83 72231 268 54 98 168 250 IFWM3303 13 79 134 157 299 32 82 168 63 IFWM330432 26 32 53 120 40 37 92 23 IFWM3305 31 114 113 154 223 17 127 201 245IFWM3291 28 45 51 122 317 60 67 125 82 IFWM3306 20 27 25 86 147 30 29 7038 IFWM3307 46 62 55 305 426 55 58 162 132 IFWM3308 38 52 53 203 246 4945 99 109 IFWM3309 55 109 140 275 383 41 120 139 96 IFWM3310 65 57 40101 177 63 54 91 384 IFWM3311 12 36 21 56 223 16 56 72 134 IFWM3312 1322 20 68 144 22 13 38 35 IFWM3313 56 74 84 221 354 74 118 155 295IFWM3314 20 26 21 39 92 16 28 42 57 IFWM3315 63 59 50 134 240 92 61 115192 IFWM3316 42 42 33 70 168 57 87 73 123 IFWM3317 16 133 121 206 370 11126 162 223 IFWM3318 18 30 34 119 252 48 40 91 64 IFWM3319 34 52 44 151274 76 49 107 152 IFWM3320 21 20 13 61 89 26 33 41 24 IFWM3321 33 33 2479 159 45 36 58 90 IFWM3322 40 42 33 127 272 56 58 86 37 IFWM3323 73 7748 144 337 77 94 121 104 IFWM3325 11 22 32 92 111 6 33 91 55 IFWM3327 3934 35 113 164 60 35 106 60 IFWM3328 108 99 91 360 410 132 72 207 482IFWM3329 59 50 51 166 258 95 61 173 532 IFWM3330 45 112 366 631 375 5882 175 38 IFWM3331 15 12 13 72 85 21 15 26 33 IFWM3332 34 31 54 135 13955 50 76 68 IFWM3333 53 65 89 607 477 137 77 258 457 IFWM3334 345 6205210 2400 744 436 941 517 86 IFWM3335 42 49 61 245 336 111 77 137 151IFWM3336 19 47 61 110 235 46 51 105 35 IFWM3337 20 17 16 71 91 22 34 3743 IFWM3338 57 46 64 245 319 108 46 200 134 IFWM3339 49 59 65 161 235 9479 141 115 IFWM3340 4 48 41 124 201 8 24 56 44 IFWM3341 8 104 88 275 4457 63 134 151 IFWM3342 68 142 164 269 589 43 14 95 47 IFWM3343 20 65 63157 237 60 60 83 106 IFWM3344 78 161 140 274 442 80 112 105 305 IFWM329218 123 98 295 613 21 49 72 254 IFWM3345 4 72 120 169 324 9 92 111 158IFWM3346 52 105 117 505 480 44 86 170 183 IFWM3347 48 96 136 411 350 4184 125 347 IFWM3348 50 83 64 164 89 58 20 49 42 IFWM3349 7 56 52 123 1748 65 102 291 IFWM3350 20 57 54 55 161 33 98 66 366 IFWM3351 4 48 53 140113 10 15 33 30 IFWM3352 60 121 152 287 267 48 90 87 193 IFWM3353 6 5455 95 110 6 16 31 90 IFWM3354 17 43 32 98 123 23 43 52 360 IFWM3355 2655 52 79 122 39 60 41 212 IFWM3356 274 IFWM3357 4 23 39 130 170 8 14 378 IFWM3358 18 54 47 223 319 62 48 123 103 IFWM3359 21 41 53 136 147 3440 51 152 IFWM3360 25 56 49 174 233 35 43 61 236 IFWM3361 30 101 135 388622 48 66 98 151 IFWM3364 13 93 109 254 269 39 80 118 151 IFWM3366 21 3027 109 122 40 30 66 433 IFWM3367 57 116 100 453 465 86 67 152 42735IFWM3368 28 62 59 181 203 62 48 103 47393 IFWM3369 81 88 190 622 344 123121 220 58 IFWM3370 10 39 39 225 155 24 21 41 42 IFWM3371 18 43 50 125100 23 50 64 81 IFWM3372 49 94 116 690 432 154 103 272 717 IFWM3374 55106 121 568 540 139 96 208 349 IFWM3375 19 22 23 45 67 33 35 31 27IFWM3376 20 34 39 140 113 37 48 62 81 IFWM3377 27 44 51 192 193 58 38120 132 IFWM3378 75 172 168 496 533 141 150 248 279 IFWM3379 15 13 13 3372 22 13 23 27 IFWM3380 25 46 32 76 88 14 70 59 157 IFWM3381 45 79 92121 171 58 53 67 37 IFWM3382 30 24 20 37 75 27 30 32 18 IFWM3383 28 6960 86 143 33 84 64 194 IFWM3293 29 25 17 62 166 12 37 46 120 IFWM3384 2629 33 68 77 13 22 17 25 IFWM3385 32 41 41 142 178 19 23 52 42 IFWM338626 31 29 87 103 16 26 35 43 IFWM3387 39 90 58 161 169 36 34 69 19IFWM3388 33 30 19 55 84 26 27 44 105 IFWM3389 17 24 9 32 98 10 33 30 69IFWM3390 10 11 11 30 75 14 10 29 26 IFWM3391 23 16 25 66 117 29 44 42 63IFWM3392 23 16 14 21 58 34 19 25 44 IFWM3393 57 49 41 96 166 70 64 78284 IFWM3394 45 63 63 77 105 123 91 49 304 IFWM3395 19 116 102 181 279 363 63 83 IFWM3396 11 5 7 37 79 48 61 68 52 IFWM3397 25 27 28 78 119 4243 62 103 IFWM3398 26 17 17 44 61 32 22 30 34 IFWM3399 30 25 21 50 88 3630 37 59 IFWM3400 33 23 22 60 135 32 52 49 68 IFWM3401 62 42 28 78 19542 67 76 604 IFWM3403 7 25 27 59 62 8 27 23 11 IFWM3405 20 22 19 36 6717 15 39 38 IFWM3406 78 37 37 119 135 39 37 63 34 IFWM3407 17 32 33 58128 13 27 60 65 IFWM3408 86 730 5690 1870 703 54 895 176 17 IFWM3409 1411 9 41 58 18 5 25 28 IFWM3410 29 37 52 82 86 59 52 52 33 IFWM3411 28 2337 151 101 53 35 87 53 IFWM3413 47 37 33 150 206 58 44 85 121 IFWM341458 68 101 174 193 13 33 70 26 IFWM3415 18 19 26 51 69 15 26 44 39IFWM3416 28 29 45 68 103 37 29 69 55 IFWM3417 29 31 39 70 100 36 30 5941 IFWM3418 89 191 229 656 1160 206 178 493 208 IFWM3419 86 126 158 399582 90 81 193 87 IFWM3420 36 113 118 299 475 61 37 124 35

Select antibodies from the panel of 126 were characterized in an ISREassay for their ability to inhibit a spectrum of IFN-α subtypes andIFN-ω, and their solubility and biophysical characteristics wereassessed. IC₅₀ values from the ISRE assay are shown in Table 11 andTable 12 for select antibodies. The IC₅₀ values were at double-digit pMor lower for several antibodies to 11 recombinant IFN-α subtypes and toIFN-ω. This represents more than a hundred-fold improvement over theparental mAb, IFWM371, whose IC₅₀ against its antigens ranging fromsingle digit to double digit nM. As the parental antibody, theaffinity-matured antibodies did not neutralize IFN-αD or IFN-β. The mostpotent affinity-matured antibody mAb IFWM3423 had almost a single-digitpicomolar IC₅₀ to all interferon subtypes it bound.

TABLE 11 IC₅₀ (pM) mAbs αA αB2 αC αD αF αG αH2 IFWM371 8400 19300 53900NN 16000 12900 9600 IFWM3304 29 39 117 NN 62 47 42 IFWM3307 73 247 583NN 170 95 88 IFWM3308 94 230 996 NT 155 167 32 IFWM3310 50 43 196 NN 11173 45 IFWM3314 29 33 157 NN 58 57 40 IFWM3320 45 18 392 NT 54 82 23IFWM3321 29 87 266 NN 54 34 28 IFWM3322 31 121 306 NN 117 83 46 IFWM3328216 520 1416 NN 631 486 440 IFWM3331 18 80 98 NN 48 27 33 IFWM3332 104327 479 NN 228 99 92 IFWM3385 63 158 272 NN 66 77 29 IFWM3399 43 62 189NN 29 29 13 IFWM3400 35 86 138 NN 43 35 15 IFWM3405 40 99 68 NN 35 26 16IFWM3410 NT 211 168 NN 77 55 33 IFWM3416 81 250 112 NN 64 49 35 IFWM342113 16 20 NN 11 18 8 IFWM3423 12 11 12 NN 8 9 4 NN: not neutralizing; NT:not tested

TABLE 12 IC₅₀ (pM) IFN- IFN- mAbs αI αJ1 αK αWA α4a β ω IFWM371 357007300 74200 32800 NN 43900 IFWM3304 157 126 57 237 112 NN 31 IFWM3307 752328 95 600 501 NN 100 IFWM3308 478 363 59 894 295 NN 40 IFWM3310 258 16696 473 169 NN 81 IFWM3314 163 86 65 188 137 NN 32 IFWM3320 355 213 34633 166 NN 53 IFWM3321 301 114 46 267 295 NN 38 IFWM3322 460 169 71 382352 NN 59 IFWM3328 2002 1657 321 3078 1169 NN 456 IFWM3331 198 94 28 109117 NN 50 IFWM3332 893 487 76 947 519 NN 228 IFWM3385 225 251 65 839 414NN 68 IFWM3399 18 106 32 189 111 NN 27 IFWM3400 137 154 35 376 220 NN 29IFWM3405 72 26 22 216 86 NN 19 IFWM3410 183 217 41 538 192 NN 89IFWM3416 158 61 43 779 201 NN 40 IFWM3421 17 14 18 14 15 NN 8 IFWM3423 46 9 10 8 NN 6 NN = not neutralizing

Example 9. Engineering of Antibodies to Minimize Post-TranslationalModification Risk

Based on neutralizing activity, solubility and biophysical properties,four mAbs derived from affinity maturation of IFWM371, IFWM3331(IFWB3066), IFWM3399 (IFWB3134), IFWM3421 (IFWB3156) and IFWM3423(IFWB3158) were analyzed further. The heavy chains of these mAbs consistof either IFWH591 (SEQ ID NO: 28) or IFWH629 (SEQ ID NO: 31) and thelight chains of them consist of either IFWL984 (SEQ ID NO: 71) orIFWL1048 (SEQ ID NO: 53) or IFWL1073 (SEQ ID NO: 61).

Both VH chains contain several potential post-translational modification(PTM) motifs in their CDRs, including an acid-catalyzed hydrolysissequence motif (D52-P53), an isomerization motif (D55-S56) on HCDR2 andpotential oxidation sites on HCDR1 (W33) and CDR-H3 (W104).

The VL of IFWL984 (SEQ ID NO: 71) and IFWL1048 (SEQ ID NO: 53) containone isomerization motif (D30-G31) on LCDR1 while the VL of IFW1073 (SEQID NO: 61) contains potential oxidation sites on LCDR3 (W92 and W94) anda potential deamidation site on LCDR1 (N31-S32).

To reduce PTM risks on heavy chain CDRs, D52 in HCDR2 was back-mutatedto the germline residue tyrosine (D52Y). P53 was mutated to Alanine.W104 in HCDR3 (VH_W104) was replaced with alanine, tyrosine, serine oraspartic acid. The mutated heavy chains were co-expressed with threedifferent light chains and tested in the ISRE assay. From theseexperiments, antibodies with heavy chain IFWH615 (SEQ ID NO: 157) andIFWH617 (SEQ ID NO: 158) were characterized further.

To reduce PTM risks on VL IFWL984 (SEQ ID NO: 71) and IFWL1048 (SEQ IDNO: 53), a series of mutations to remove the potential PTM motifs weredesigned with the guidance of the structural information obtained fromthe IFWM371/IFN-ω complex structure described in Example 6. In addition,to improve the solubility of IFWM3421 (IFWB3156) and IFWM3423 (IFWB3158)having the common light chain IFWL984, a series of mutations on severalhydrophobic residues in their CDRs were made to decrease the overallsurface hydrophobicity of the antibody light chains. The IFWL984variants were expressed in HEK293E cells with the parental heavy chainIFWH591 and the expressed antibody in cell supernatants were screened inthe ISRE reporter gene assay for inhibition of IFN-ω and leukocyte IFNusing methods described in example 11. The resulting antibodies IFWB3196(D30E F32Y), IFWB3201 (D30S, G31S), and IFWB3202 (D30S, G31S, F32Y)retained good neutralizing activity. Table 13 shows the VL sequences ofthe generated antibodies having the parental IFWH591 heavy chainvariable region (SEQ ID NO: 28) and a variant IFWL984 light chain. Theparental IFWM3421 has the IFWH591 VH and the parental IFWM3423 has theIFWH629 VH. Table 14 shows the IC₅₀ values for neutralization of IFN-ωand leukocyte IFN of select generated antibodies.

TABLE 13 Antibody Antibody VL Peptide VL SEQ DNA ID AA ID ID VLsubstitution ID NO: IFWM3421 IFWB3156 IFWL984 Parent control  71IFWM3423 IFWB3158 IFWL984 Parent control  71 IFWM3454 IFWB3189 IFWL1112IFWL984 D30S 123 IFWM3514 IFWB3248 IFWL1113 IFWL984 D30E 124 IFWM3455IFWB3190 IFWL1114 IFWL984 F32Y 125 IFWM3456 IFWB3191 IFWL1115 IFWL984F50A 126 IFWM3458 IFWB3193 IFWL1117 IFWL984 F50I 127 IFWM3459 IFWB3194IFWL1118 IFWL984 F50L 128 IFWM3460 IFWB3195 IFWL1119 IFWL984 F50V 129IFWM3461 IFWB3196^(i) IFWL1120 IFWL984 D30E, F32Y 130 IFWM3462 IFWB3197IFWL1121 IFWL984 D30E, F50A 131 IFWM3463 IFWB3198 IFWL1122 IFWL984 D30E,F50I 132 IFWM3464 IFWB3199 IFWL1123 IFWL984 D30E, F50L 133 IFWM3465IFWB3200 IFWL1124 IFWL984 D30E, F50V 134 IFWM3466 IFWB3201 IFWL1125IFWL984 D30S, G31S 135 IFWM3467 IFWB3202 IFWL1126 IFWL984 136 D30S,G31S, F32Y IFWM3470 IFWB3205 IFWL1129 IFWL984 137 D30G, G31D, F50YIFWL1173 IFWL984 138 D30E, F32Y, F50Y IFWM3526 IFWB3251 IFWL1174 IFWL984D30E, F50Y 139 IFWL1175 IFWL984 140 D30S, G31S, F50Y

TABLE 14 IC₅₀ (pM) Antibody Antibody Leukocyte DNA ID AA ID human IFN-ωIFN IFWM3421 IFWB3156 7.4  0.7 IFWM3423 IFWB3158 10.7  1.1 IFWM3454IFWB3189 11.2  1.3 IFWM3514 IFWB3248 10.4  1.4 IFWM3455 IFWB3190 24 notfitted IFWM3456 IFWB3191 100.3 22.6 IFWM3458 IFWB3193 37  2.1 IFWM3459IFWB3194 1518.9  4.4 IFWM3460 IFWB3195 43.5 not fitted IFWM3461IFWB3196^(i) 21  1   IFWM3462 IFWB3197 41.6 12.6 IFWM3463 IFWB3198 41.4 6.6 IFWM3464 IFWB3199 46.8  5.6 IFWM3465 IFWB3200 33.7  1.7 IFWM3466IFWB3201 10.3  0.9 IFWM3467 IFWB3202 51.8  0.8 IFWM3470 IFWB3205 52.3 7.4

Similarly, 26 IFWL1048 variants were constructed to reduce the PTMrisks. The generated light chains were co-expressed with a heavy chainIFWH591 in HEK293E cells and the supernatant containing the antibodyscreened with ISRE assay. Table 15 shows the VH and VL sequences of thegenerated antibodies, and Table 16 shows the IC₅₀ values of theantibodies for IFN-ω and leukocyte IFN. The resulting antibodies withvariant IFWL1048 chains where the DG motif (D30-G31) in LCDR1 waseliminated, including IFWB3210 (D305), IFWB3211 (D30E) and IFWB3223(D30S, G315), showed similar neutralization activity as the parent mAbs,IFWB3056 (VL: IFWL1048, VH: IFWH591) and IFWB3134 (VL: IFWL1048; VH:IFWH629). However, resulting antibodies with variant IFWL1048 chainswith the DG motif eliminated and substitutions made to reducehydrophobicity, including IFWB3219 (D30E, A32Y), IFWB3227 (D30S, G315,F94L) and IFWB3230 (D30S, G31S, A32Y, F94L) demonstrated a loweractivity than the parental mAbs.

TABLE 15 Antibody Antibody VL Peptide VL SEQ DNA ID AA ID ID Mutation IDNO: IFWM3321 IFWB3056 IFWL1048 Parent control  53 IFWM3399 IFWB3134IFWL1048 Parent control  53 IFWM3475 IFWB3210 IFWL1135 IFWL1048 D30S 141IFWM3476 IFWB3211 IFWL1136 IFWL1048 D30E  73 IFWM3477 IFWB3212 IFWL1137IFWL1048 A32Y 142 IFWM3483 IFWB3218 IFWL1143 IFWL1048 F94L 143 IFWM3484IFWB3219 IFWL1144 IFWL1048 D30E, A32Y  74 IFWM3488 IFWB3223 IFWL1148IFWL1048 D30S, G31S  75 IFWM3489 IFWB3224 IFWL1149 IFWL1048 D305, G315,144 A32Y IFWM3492 IFWB3227 IFWL1152 IFWL1048 D305, G315, 145 F94LIFWM3495 IFWB3230 IFWL1155 IFWL1048 D305, G315, 146 A32Y, F94L IFWL1161IFWL1048 D30G, G31D, 147 A32F, F50A, F94L

TABLE 16 IC₅₀ (pM) Antibody Antibody human Leukocyte DNA ID AA ID IFN-ωIFN IFWM3321 IFWB3056 25.8 1.2 IFWM3399 IFWB3134 28.7 1.3 IFWM3475IFWB3210 27.3 1.5 IFWM3476 IFWB3211 17.2 3.3 IFWM3477 IFWB3212 31   4.1IFWM3483 IFWB3218 51.4 4.8 IFWM3484 IFWB3219 38.7 2.3 IFWM3488 IFWB322329.4 1   IFWM3489 IFWB3224 80.3 3.4 IFWM3492 IFWB3227 56.1 0.5 IFWM3495IFWB3230 57.5 3.9

Potential PTM motifs on the VL IFWL1073 of IFWB3066 included potentialoxidation sites on LCDR3 (W92 and W94). The LCDR3 of IFWL1073(QQGWDWPLT; SEQ ID NO: 98) was replaced with a consensus LCDR3 sequenceidentified present in the LCDR3 of many affinity-matured antibodies(QQSYDFPLT; SEQ ID NO: 154). In addition, several mutants were designedto address a potential deamidation site (N31-S32) on LCDR1. 14 generatedvariants of IFWL1073 were paired with IFWH591 and expressed in 48-wellHEK293E transient transfection. The cell supernatants were testeddirectly in ISRE assay for their neutralization activities againstrecombinant human IFN-ω and viral-induced leukocytes expressed IFNs. ThemAbs with mutations W93Y and/or W95F showed some improvements inneutralization activity. Mutants to remove the NS motif by substitutionor by shortening CDR-L1 showed reduction or loss of neutralizationactivity. Table 17 shows the VH and the VL sequences of the generatedantibodies and Table 18 shows the IC₅₀ values for IFN-ω and leukocyteIFN.

TABLE 17 Antibody Antibody VL Peptide VL SEQ DNA ID AA ID ID Mutation IDNO: IFWM3331 IFWB3066 IFWL1073 Parent control  61 IFWM3501 IFWB3236IFWL1162 IFWL1073 W93Y 148 IFWM3502 IFWB3237 IFWL1163 IFWL1073 W95F 149IFWM3503 IFWB3238 IFWL1164 IFWL1073 W93Y, W95F 150 IFWM3527 IFWB3252IFWL1176 IFWL1073 151 N31Q, W93Y, W95F IFWM3528 IFWB3253 IFWL1177IFWL1073 152 N31T, W93Y, W95F IFWM3529 IFWB3254 IFWL1178 IFWL1073 153S32T, W93Y, W95F

TABLE 18 IC 50 (pM) Antibody Antibody human Leukocyte DNA ID AA ID IFN-ωIFN IFWM3331 IFWB3066 40.7 1.5 IFWM3501 IFWB3236 15.1 2.6 IFWM3502IFWB3237 28 2.5 IFWM3503 IFWB3238 12 2.1

Select VL variants derived from the engineering efforts to minimize thePTM risk were paired with either IFWH591 or IFWH629 and scaled up forexpression and purification. Table 19 shows the VL/VH pairing of theantibodies. Table 20 shows the IC₅₀ values of the select resultingantibodies for various recombinant IFN-α subtypes and IFN-ω.

TABLE 19 VL HC VL VH SEQ SEQ Antibody Peptide ID ID NO: DescriptionPeptide ID ID NO: IFWL1073 IFWL1073 61 parent IFWH591 28 mutantsIFWL1164 150 IFWL1073 IFWH591 28 W93Y, W95F IFWL1176 151 IFWL1073 N31Q,IFWH591 28 W93Y, W95F IFWL1177 152 IFWL1073 N31T, IFWH591 28 W93Y, W95FIFWL984 IFWL984 71 parent IFWH591 28 mutants IFWL984 71 parent IFWH62931 IFWL984 71 parent IFWH629 31 IFWL1125 135 IFWL984 IFWH591 28 D30S,G31S IFWL1126 136 IFWL984 D305, IFWH591 28 G31S, F32Y IFWL1174 139IFWL984 IFWH591 28 D30E, F50Y IFWL1048 53 parent IFWH591 28 IFWL1048 53IFWH629 31 IFWL1136 73 IFWL1048 D30E IFWH591 28 IFWL1148 75 IFWL1048IFWH591 28 D30S, G31S

TABLE 20 Protein Protein AA (IC₅₀ (pM) DNA ID ID αA αB2 αC αF αG α4a ωIFWM3331* IFWB3066 26 77 48 157 33 52 31 65 88 30 23 30 IFWM3421*IFWB3156 16 19 29 16 18 22 8 17 19 28 15 14 19 9 IFWM3423* IFWB3158 2017 24 21 20 23 16 12 13 17 11 10 9 7 IFWM3466 IFWB3201 17 21 31 18 16 2312 IFWM3503 IFWB3238 15 19 20 20 15 32 13 IFWM3399 IFWB3134 24 38 64 2924 117 25 IFWM3476 IFWB3211 35 54 115 50 39 130 23 IFWM3488 IFWB3223 1931 72 25 21 84 22 *results from two independent experiments

Example 10. Broad Neutralizing Ability of Anti-IFN-α/ω Antibodies

Several of the generated antibodies neutralized IFN-ω and multiple IFN-αsubtypes with an IC₅₀ of 100 pM or less, measured using the ISRE assaydescribed above. The variable region sequences of these antibodies areshown in Table 21. Table 22 shows the LCDR1 sequences, Table 23 theLCDR2, Table 24 the LCDR3, Table 25 the HCDR1, Table 26 the HCDR2 andTable 27 the HCDR3 of the antibodies. FIG. 10 shows the IC₅₀ values foreach Type I IFN in the ISRE assay.

TABLE 21 VH SEQ VL SEQ mAbs cDNA mAb protein VH ID NO: VL ID NO:IFWM3308 IFWB3043 IFWH591 28 IFWL1004 40 IFWM3307 IFWB3042 IFWH591 28IFWL1001 39 IFWM3410 IFWB3145 IFWH629 31 IFWL1074 62 IFWM3322 IFWB3057IFWH591 28 IFWL1051 54 IFWM3385 IFWB3120 IFWH629 31 IFWL1001 39 IFWM3416IFWB3151 IFWH629 31 IFWL1091 68 IFWM3310 IFWB3045 IFWH591 28 IFWL1007 42IFWM3400 IFWB3135 IFWH629 31 IFWL1051 54 IFWM3321 IFWB3056 IFWH591 28IFWL1048 53 IFWM3522 IFWB3211 IFWH591 28 IFWL1136 73 IFWM3524 IFWB3223IFWH591 28 IFWL1148 75 IFWM3320 IFWB3055 IFWH591 28 IFWL1047 52 IFWM3304IFWB3039 IFWH591 28 IFWL997 35 IFWM3520 IFWB3201 IFWH591 28 IFWL1125 135IFWM3399 IFWB3134 IFWH629 31 IFWL1048 53 IFWM3314 IFWB3049 IFWH591 28IFWL1014 46 IFWM3331 IFWB3066 IFWH591 28 IFWL1073 61 IFWM3405 IFWB3140IFWH629 31 IFWL1063 57 IFWM3442 IFWB3177 IFWH615 157 IFWL984 71 IFWM3525IFWB3238 IFWH591 28 IFWL1164 150 IFWM3423 IFWB3158 IFWH629 31 IFWL984 71IFWM3444 IFWB3179 IFWH617 158 IFWL984 71 IFWM3421 IFWB3156 IFWH591 28IFWL984 71

TABLE 22 LCDR1 mAbs Sequence SEQ ID NO: IFWM3308 Q S I A E F 77 IFWM3307Q S I G D F 85 IFWM3410 Q S I A N T N 79 IFWM3322 Q S I A D F 76IFWM3385 Q S I G D F 85 IFWM3416 Q S I R N T N 89 IFWM3310 Q S I G K S86 IFWM3400 Q S I A D F 76 IFWM3321 Q S I D G A 80 IFWM3522 Q S I E G A84 IFWM3524 Q S I S S A 90 IFWM3320 Q S I N G V 88 IFWM3304 Q S I G S A87 IFWM3520 Q S I S S F 91 IFWM3399 Q S I D G A 80 IFWM3314 Q S I D R A83 IFWM3331 Q S I D N S Y 82 IFWM3405 Q S I A N N N 78 IFWM3442 Q S I DG F 81 IFWM3525 Q S I D N S Y 82 IFWM3423 Q S I D G F 81 IFWM3444 Q S ID G F 81 IFWM3421 Q S I D G F 81

TABLE 23 LCDR2 mAbs Sequence SEQ ID NO: IFWM3308 F A S 93 IFWM3307 F A S93 IFWM3410 W A S 95 IFWM3322 F A S 93 IFWM3385 F A S 93 IFWM3416 W A S95 IFWM3310 F A S 93 IFWM3400 F A S 93 IFWM3321 F A S 93 IFWM3522 F A S93 IFWM3524 F A S 93 IFWM3320 F A S 93 IFWM3304 F A S 93 IFWM3520 F A S93 IFWM3399 F A S 93 IFWM3314 F A S 93 IFWM3331 G A S 94 IFWM3405 W A S95 IFWM3442 F A S 93 IFWM3525 G A S 94 IFWM3423 F A S 93 IFWM3444 F A S93 IFWM3421 F A S 93

TABLE 24 LCDR3 SEQ ID mAbs Sequence NO: IFWM3308 Q Q S I D F P L T 104IFWM3307 Q Q A L D F P L T 96 IFWM3410 Q Q W Y D N P L T 107 IFWM3322 QQ S H S F P L T 103 IFWM3385 Q Q A L D F P L T 96 IFWM3416 Q Q G Y D T PF T 100 IFWM3310 Q Q S Y D F P L T 105 IFWM3400 Q Q S H S F P L T 103IFWM3321 Q Q A Y D F P L T 97 IFWM3522 Q Q A Y D F P L T 97 IFWM3524 Q QA Y D F P L T 97 IFWM3320 Q Q S H D F P L T 102 IFWM3304 Q Q S Y D F P LT 105 IFWM3520 Q Q S Y D L P I T 106 IFWM3399 Q Q A Y D F P L T 97IFWM3314 Q Q S F D F P L T 101 IFWM3331 Q Q G W D W P L T 98 IFWM3405 QQ G Y D T P F T 100 IFWM3442 Q Q S Y D L P I T 106 IFWM3525 Q Q G Y D FP L T 99 IFWM3423 Q Q S Y D L P I T 106 IFWM3444 Q Q S Y D L P I T 106IFWM3421 Q Q S Y D L P I T 106

TABLE 25 HCDR1 mAbs Sequence SEQ ID NO: IFWM3308 G Y S F T S Y W 109IFWM3307 G Y S F T S Y w 109 IFWM3410 G Y S F T S Y W 109 IFWM3322 G Y SF T S Y W 109 IFWM3385 G Y S F T S Y W 109 IFWM3416 G Y S F T S Y W 109IFWM3310 G Y S F T S Y W 109 IFWM3400 G Y S F T S Y W 109 IFWM3321 G Y SF T S Y W 109 IFWM3522 G Y S F T S Y W 109 IFWM3524 G Y S F T S Y W 109IFWM3320 G Y S F T S Y W 109 IFWM3304 G Y S F T S Y W 109 IFWM3520 G Y SF T S Y W 109 IFWM3399 G Y S F T S Y W 109 IFWM3314 G Y S F T S Y W 109IFWM3331 G Y S F T S Y W 109 IFWM3405 G Y S F T S Y W 109 IFWM3442 G Y SF T S Y W 109 IFWM3525 G Y S F T S Y W 109 IFWM3423 G Y S F T S Y W 109IFWM3444 G Y S F T S Y W 109 IFWM3421 G Y S F T S Y W 109

TABLE 26 HCDR2 mAbs Sequence SEQ ID NO: IFWM3308 I D P S D S D T 113IFWM3307 I D P S D S D T 113 IFWM3410 I D P S D S D T 113 IFWM3322 I D PS D S D T 113 IFWM3385 I D P S D S D T 113 IFWM3416 I D P S D S D T 113IFWM3310 I D P S D S D T 113 IFWM3400 I D P S D S D T 113 IFWM3321 I D PS D S D T 113 IFWM3522 I D P S D S D T 113 IFWM3524 I D P S D S D T 113IFWM3320 I D P S D S D T 113 IFWM3304 I D P S D S D T 113 IFWM3520 I D PS D S D T 113 IFWM3399 I D P S D S D T 113 IFWM3314 I D P S D S D T 113IFWM3331 I D P S D S D T 113 IFWM3405 I D P S D S D T 113 IFWM3442 I A PS D S D T 111 IFWM3525 I D P S D S D T 113 IFWM3423 I D P S D S D T 113IFWM3444 I D A S D S D T 112 IFWM3421 I D P S D S D T 113

TABLE 27 HCDR3 mAbs Sequence SEQ ID NO: IFWM3308 A R H P G L N W A P D FD Y 116 IFWM3307 A R H P G L N W A P D F D Y 116 IFWM3410 A R H P G L AW A P D F D Y 115 IFWM3322 A R H P G L N W A P D F D Y 116 IFWM3385 A RH P G L A W A P D F D Y 115 IFWM3416 A R H P G L A W A P D F D Y 115IFWM3310 A R H P G L N W A P D F D Y 116 IFWM3400 A R H P G L A W A P DF D Y 115 IFWM3321 A R H P G L N W A P D F D Y 116 IFWM3522 A R H P G LN W A P D F D Y 116 IFWM3524 A R H P G L N W A P D F D Y 116 IFWM3320 AR H P G L N W A P D F D Y 116 IFWM3304 A R H P G L N W A P D F D Y 116IFWM3520 A R H P G L N W A P D F D Y 116 IFWM3399 A R H P G L A W A P DF D Y 115 IFWM3314 A R H P G L N W A P D F D Y 116 IFWM3331 A R H P G LN W A P D F D Y 116 IFWM3405 A R H P G L A W A P D F D Y 115 IFWM3442 AR H P G L N W A P D F D Y 116 IFWM3525 A R H P G L N W A P D F D Y 116IFWM3423 A R H P G L A W A P D F D Y 115 IFWM3444 A R H P G L N W A P DF D Y 116 IFWM3421 A R H P G L N W A P D F D Y 116

Example 11. Anti-IFN-α/ω Antibodies Neutralize Leukocyte IFN

The ability of the antibodies to neutralize leukocyte IFN was assessedby the ability of the antibodies to inhibit IFN-induced IP-10 releasefrom whole blood.

Select antibodies from the affinity-maturation campaign or afterminimizing the PTM risk were characterized further for their ability toinhibit endogenous Type I IFN. All characterized antibodies were ofIgG1/K type. Antibodies IFWM3522, IFWM3525, IFWM3399 and IFWM3423 wereused in the assays.

IP-10 Release Assay:

240 μl of whole blood (Biological Specialty Corporation) was added toindividual wells in 96 well U-bottom plates containing 30 μl of antibody(anti IFN-α/ω or isotype control), with or without IFN or IFN-containingconditioned media diluted in cell culture media (RPMI1640 with 10% HIFBS and 1% penn strep). For stimulation, human leukocyte IFN(Sigma-Aldrich) was utilized at 250 U/ml (final volume) and SLE immunecomplex-treated conditioned media at 10 μl per well. IFN and antibodymixtures were preincubated at room temperature for 20-30 min prior toadding whole blood. Plates were incubated overnight for 20-22 hours at37° C. The following day, plates were centrifuged at 400×g for 5 minutesat room temperature and plasma removed and frozen at −20° C. Duplicatesamples from each treatment were analyzed using a CXCL10/IP-10 ELISA kitfrom Qiagen. Upon thawing, the collected plasma was diluted 2.5 foldusing sample dilution buffer and used in the assay. Manufacturer'sprotocol was followed with slight modification in the dilution ofstandards as follows. Two fold serial dilutions of the antigen standardwere made starting at a concentration of 4000 pg/ml and ending at 31.25pg/ml. Plates were read at an absorbance at 450 nm within 30 minutes ofstopping the reaction. Analysis was performed using Softmax Pro.

Results

Select antibodies were characterized for their ability to neutralizeendogenous IFN-I preparations in relevant cell types. IFN-I stimulationof whole blood induces IP-10 (CXCL10) release in vitro and in vivo(Arico, E. et al. Concomitant detection of IFNalpha signature andactivated monocyte/dendritic cell precursors in the peripheral blood ofIFNalpha-treated subjects at early times after repeated local cytokinetreatments. J Transl Med 9, 67, doi:10.1186/1479-5876-9-67 (2011).Mohty, A. M. et al. Induction of IP-10/CXCL10 secretion as animmunomodulatory effect of low-dose adjuvant interferon-alpha duringtreatment of melanoma. Immunobiology 215, 113-123,doi:10.1016/j.imbio.2009.03.008 (2010)). IP-10 is elevated in SLE, andhas been shown in several studies to correlate with disease activity andclinical manifestations of disease (Bauer, J. W. et al.Interferon-regulated chemokines as biomarkers of systemic lupuserythematosus disease activity: a validation study. Arthritis andrheumatism 60, 3098-3107, doi:10.1002/art.24803 (2009). Kong, K. O. etal. Enhanced expression of interferon-inducible protein-10 correlateswith disease activity and clinical manifestations in systemic lupuserythematosus. Clinical and experimental immunology 156, 134-140,doi:10.1111/j 0.1365-2249.2009.03880.x (2009). Rose, T. et al. IFNalphaand its response proteins, IP-10 and SIGLEC-1, are biomarkers of diseaseactivity in systemic lupus erythematosus. Annals of the rheumaticdiseases 72, 1639-1645, doi:10.1136/annrheumdis-2012-201586 (2013).

The ability of anti IFN-α/ω mAbs to inhibit IP-10 release in whole bloodinduced by leukocyte IFN was examined in vitro. IFN-I is rapidlyproduced in response to infectious agents, such as viruses, to helpcontrol infection. Human leukocyte IFN is a natural mixture of IFNsproduced by leukocytes after viral infection and is largely composed ofIFN-α subtypes and IFN-ω. IFN-ω) is believed to constitute approximately15% of the total IFN-I activity in these preparations. Importantly,infections are believed to potentially contribute to both induction andexacerbation of SLE. In this study, human leukocyte IFN was added towhole blood samples from 2 healthy human donors in the presence ofinhibitors or controls and plasma was assessed for IP-10 release 24 hpost IFN exposure. Anti IFN-α/ω mAbs: IFWM3522 and IFWM3525 (FIG. 11A),and IFWM3399 (FIG. 11B) all dose-dependently neutralized leukocyteIFN-induced IP-10 release in both donors tested.

Example 12. Anti-IFN-α/ω Antibodies Neutralize SLE Immune Complexes

A hallmark of SLE is the presence of autoantibodies such asanti-double-stranded DNA (anti-dsDNA) that typically precede thedevelopment of clinically defined disease. Autoantibodies bound tonucleic acid ligands are thought to be endogenous inducers of type I IFNin SLE patients. The preponderance of autoantibodies in conjunction withimpaired clearance of autoantigens leads to a feedback cycle of IFNproduction where Fc receptor-dependent internalization of immunecomplexes into plasmacytoid dendritic cells (pDC) leads to increasedamounts of circulating IFN and establishment of the IFN gene signature.

We further tested the ability of the anti-IFN-α/ω antibodies toneutralize more disease relevant endogenous IFN preparations.

Immune complexes were prepared essentially as described in Example 1.These SLE patient-derived immune complexes were then added to healthydonor PBMCs and IFN-containing conditioned media collected from cellcultures (IC92 and IC163). Next, the conditioned media was added tohealthy donor whole blood from 4 healthy donors in the presence ofinhibitors or control to determine the impact of IFN-α/ω neutralizationon IFN-induced IP-10 release. IFWM3522, IFWM3525, and IFWM3399 alldose-dependently neutralized IP-10 release using both SLE immunecomplex-induced IFN preps in all whole blood donors tested. FIG. 12Ashows neutralization of SLE immune complex-induced IFN-stimulated IP-10release in human whole blood by antibodies IFWM3522 and IFWM2525 fromone donor (SLE donor 92). FIG. 12B shows the results for antibodyIFWM3399 and isotype control.

Example 13. Anti-IFN-α/ω Antibodies Neutralize SLE Plasma

Anti IFN-α/ω mAbs demonstrated potent dose-dependent neutralization ofendogenous IFN-I preparations produced from human primary cells afterexposure to both sterile (immune complex; Example 12) and microbialligands (leukocyte IFN; Example 11). Potency of the IFN-α/ω mAbs toneutralize physiological Type I IFN was further assessed by the abilityof the antibodies to neutralize IFN-I activity from SLE patient sera andplasma. This approach thus assesses ability of the antibodies toneutralize the actual circulating IFN-I milieu from the patient whichmay contain an IFN spectrum that may be difficult to recapitulate invitro.

ISRE Assay Using SLE Serum:

HEK Blue (α/β) cells (InvivoGen) were plated at 50,000 cells per well ina total volume of 200 μl DMEM+10% FBS and incubated overnight at 37° C.The next day, pooled plasma (3 donors) or serum (13 donors) pre-selectedon the basis of achieving an OD of greater than or equal to 1.0 after a30 minute incubation in this assay was thawed and mixed at a 1:1 (v/v)ratio with DMEM+10% FBS. Supernatants were removed from the previouslyplated Hek Blue cells and replaced with 100 μl of the SLE plasma orserum/media mixture and allowed to incubate overnight at 37° C. The nextday, 40 μl of conditioned media was removed and added to 160 μlQuanti-Blue substrate (InvivoGen) in a new plate and allowed to incubatefor 30 minutes. Plates were read using a spectrophotometer at 650nanometer wavelength and IC₅₀ values were calculated using GraphPadPrism.

Results

SLE serum from a Chinese cohort of patients (SLE Cohort 1) and SLEplasma from a primarily African American cohort (SLE Cohort 2) wasprescreened for IFN-I activity using the ISRE assay. SLE donor serum orplasma samples having an OD of ˜1.0 or greater were determined to have asufficient window of IFN-I activity such that inhibition with antagonistantibodies could be easily measured. These donor samples were thenpooled to create a serum or plasma stock to generate enough samplevolume to enable repeat experiments and antibody titrations. SLE patientsamples from diverse racial/ethnic cohorts were utilized to bettercapture the potential diversity in qualitative and quantitative IFN-Iresponses in SLE patients. African American and Asian donors are thoughtto have higher IFN-I activity than Caucasian donors. The anti-IFN-α/ωmAbs tested dose-dependently neutralized IFN-I activity in pooled SLEpatient serum and plasma samples. IC₅₀ values from two independentexperiments are shown using pooled samples from both SLE cohorts inTable 28.

TABLE 28 Mean IC₅₀ (ng/ml) +/− SD mAb SLE Cohort 1 (serum) SLE Cohort 2(plasma) IFWM3525 5.166 +/− 0.1612 4.255 +/− 0.8422 IFWM3522 10.47 +/−0.3818 6.059 +/− 0.3613 IFWM3399 8.352 +/− 1.102  4.340 +/− 0.1223

Example 14. Anti-IFN-α/ω Antibodies Neutralize IFN Gene Signature

Type I IFN induces a spectrum of genes that are also overexpressed insome SLE patients as compared to healthy controls. Plasma samples fromSLE patients exhibiting this IFN gene signature are capable of inducingoverexpression of a similar set of genes when added to healthy donorPBMCs or cell lines, and this activity is predominately neutralized byantibodies targeting IFN-α (Hua et al., Arthritis and rheumatism 54,1906-1916, doi:10.1002/art.21890 (2006)).

An assay was developed to determine the effect of the antibodies onnormalizing the IFN-I signature present in the SLE patient heparinizedwhole blood. IFN-I inducible gene MX1 (myxovirus resistance 1)expression was used as a marker for IFN-I activity.

Materials

2-4 h after collection of SLE or healthy blood into sodium heparintubes, 240 μl was plated into 96 well U-bottom plates containinganti-IFN-α/ω antibodies or human IgG1 isotype control. Antibodiesdiluted in PBS were added at 30 μl per well to 240 μl of blood. After 24h incubation at 37° C., 745 μl of PAXgene stabilization reagent (QIAGEN)was added to a 96 deep well plate and blood samples were transferred andmixed thoroughly by pipetting. Plates were sealed and frozen at −80° C.until further processing. After thawing, samples were transferred to 2ml Safe-Lock tubes (Eppendorf) and spun at 5000×g for 10 minutes.Supernatants were aspirated and sample pellets resuspended in 432 μl ofDNase/RNase free water by vortexing. Samples were further centrifuged at5000×g and pellets resuspended in 350 μl BR1 buffer. 300 μl of BR2buffer was next added followed by 40 μl of proteinase K and samplesincubated at 55° C. and shaken at 800 rpm for 10 minutes. Themanufacturer's protocol was followed for remainder of purification(QIAGEN, cat#762164). 120 ng of total RNA from each sample was convertedto cDNA using iScript cDNA Synthesis kit (BIO-RAD) and primer/probepairs for human MX1 and beta actin (ACTB) (cat# Hs00895608_m1 andHs01060665_g1, respectively) were utilized for qPCR. Data was collectedon a Viia7 Real Time PCR system and analyzed us GraphPad Prismrepresenting the change in expression of MX1 relative to the ACTB (dCT).

Results

The ability of the IFN-α/ω antibodies to decrease the IFN-I signature inpatient blood was assessed using MX1 gene expression as a marker forIFN-I activity.

MX1 gene expression was increased approximately 7 fold in the blood of aSLE patient when compared to a healthy control. The tested anti-IFN-α/ωantibodies dose-dependently reduced MX1 expression in the blood of SLEpatients after 24 hour incubation, and at highest antibody concentrationthe MX1 expression was normalized close to the levels observed inhealthy control. FIG. 13 shows the effect of the antibody treatment onMX1 expression in one SLE donor normalized to beta actin expression andis representative of multiple donors having elevated baseline MX1expression when compared to healthy controls.

Example 15. Anti-IFN-α/ω Antibodies Neutralize Cyno Type I IFNs

The ability of the select anti-IFN-α/ω antibodies to neutralize variouscyno Type I IFNs was assessed using the ISRE reporter gene assay.

Cynomolgus IFN-α2 (PBL Assay Sciences), IFN-α4 (Sino Biological), IFN-α8(Sino Biological), and IFN-α13 (Sino Biological) were used in theassays. IC₅₀ values were determined using previously determined EC₇₅values for each IFN. (0.078 ng/ml for IFN-α2, 2.68 ng/ml for IFN-α4,0.66 ng/ml for IFN-α8 and 18.4 for IFN-α13). The IC₅₀ of selectanti-IFN-α/ω mAbs is shown in Table 29). The data in table 20 is anaverage of two independent experiments. IFN-α/ω mAbs IFWM3525 andIFWM3522 exhibited similar cross-neutralization properties between thehuman and orthologous cynomolgus antigens available to test. The lack ofneutralization of cynomolgus IFN-α13 was expected, as this molecule,like human IFN-αD, has a serine at position 27 (S27).

TABLE 29 Mean IC₅₀ (ng/ml) +/− SD Cyno Cyno Cyno Cyno Cyno mAb IFN-α13IFN-α4 IFN-α2 IFN-α8 IFN-ω IFWM3525 921.5 +/− 6.769 +/− 3.346 +/− 0.5668+/− 0.9568 +/− 294.9 0.1923 0.1747 0.07085 0.1276 IFWM3522 8063 +/−7.348 +/− 9.887 +/− 0.5497 +/− 2.028 +/− 2562 0.7616 2.918 0.037340.3691

Example 16. Crystal Structure of IFWM3421 in Complex with IFN-ω T80E

Crystallization, X-ray data collection and structure determination wasdone essentially as described in Example 6, except for followingchanges:

The complex was prepared by mixing IFN-ω:Fab at 1.05:1.00 ratio (excessIFN-ω), incubated at 4° C. overnight, and then concentrated withoutpurification to 8.37 mg/mL in 20 mM Tris pH 7.4, 50 mM NaCl. Crystalsfor X-ray data collection were obtained from HEPES pH 7.5, 0.2 M Li2SO4,18% PEG 3350 with MMS seeding.

For X-ray data collection for the IFNω/Fab3186 complex, a crystal wassoaked in synthetic mother liquor (0.1 M HEPES, pH 7.5, 20% PEG 3350,0.2 M LiSO4 with 20% glycerol) and flash frozen in liquid nitrogen.X-ray data were collected at APS (Argonne National Lab). ELNATeplyak-2013-0014. The diffraction data were processed with XDS. Thestructure refinement statistics are given in Table 30.

TABLE 30 IFNω/FabM3421 Crystal data Space group C2 Unit cell dimensionsa, b, c (Å) 77.48, 69.89, 127.38 α, β, γ (°) 90, 102.39, 90 Asymmetricunit content 1 complex X-ray data Resolution (Å) 50.00-1.90 (1.94-1.90)Number of measured reflections 160,439 (10,287) Number of uniquereflections 49,423 (3,356) Completeness (%) 98.3 (91.1) R_(merge) 0.095(0.643) <I/σ> 9.8 (2.5) B-factor (Wilson plot) (Å²) 26.6 RefinementResolution (Å) 44.36-1.90 (1.94-1.90) Number of refls used in refinement49,411 (2,292) Number of all atoms 4,249 Number of water molecules 442Rcryst (%) 19.0 (42.1) Rfree (%) 22.8 (39.8) RMSD bond lengths (Å) 0.008RMSD bond angles (°) 1.12 RMSD B-factor main-chain (Å²) 4.9 MeanB-factor (Å²) 34.1 Protein 33.6 Solvent 38.3 MolProbity [25] Clash score3.2 Rotamer outliers (%) 2.5 Ramachandran favored (%) 98.2 Ramachandranoutliers (%) 0.0 Cβ deviation >0.25 Å 0 *Values for high-resolutionshell are in parentheses

The crystal structure of IFNω/Fab3421 was determined to 1.9 Å (Table30). The IFN-ω model contained residues of 23-39 and 118-153. Themajority of IFN-ω molecule did not have any electron density and therewas no room for them in the crystal, suggesting that cleavage of IFN-ωalso happened.

The overall structure of the IFN-ω/Fab3421 complex was very similar toIFNω/FabM371. The backbone structures of the individual components (VH,VL and IFNω) are all nearly identical (Cα rmsd 0.17, 0.23 and 0.36 Å,respectively).

There were, however, a number of significant structural differences.First, when the two structures were superimposed on the VL, the VH wasrotated by 4 degrees and the antigen rotated by 11 degrees, leading to alarge shift of the IFN-ω molecule with respect to VL. Second, H bondingand water structures (WC2 in particular) were different between the twostructures (FIGS. 14A and 14B). R33 of IFN-ω makes 6H bonds including asalt-bridge with D107 of HCDR3 in the parent M371 complex (FIG. 14A). Inthe matured form, the side chain electron density for both R33 of IFN-ωand D107 of VH is less well defined (not shown) and they appear to befarther apart, thus reducing the number and strength of thecharge-charge interactions (FIG. 14B). A water molecule that involvesH99 of VH in M371 is now absent (FIGS. 14A, 14B). Third, F108 of HCDR3is not involved in antigen binding, but is part of the VL/VH interface.It adopts two alternative conformations in the parent structure (FIG.14C). The relative rotation of the VL/VH domains along with the L961mutation in VL reduced it to a single rotamer. Thus it appears that partof the maturation mutations led to better pairing of the Fv. Fourth, twopositions were mutated to F (A50F and Y32F) during maturation. Y32 formstwo H bonds with the backbone of IFN-ω. But these were also lost as aresult of mutation to F (FIG. 14D). The A50F mutation does not generateany new contact with the antigen. Rather its phenyl ring stacks with theVH W104, which in turn packs with the antigen (FIG. 14D). In the LCDR3,two additional hydrophobic mutations (T94L and L961) appear to formbetter hydrophobic pocket for L30 and F27 of the antigen. Two additionalnegative charge mutations (S39D and S93D) do not form any interactions,except with solvent. Overall, affinity improvement is the result of thematuration process that reduces polar interactions butfavors/strengthens hydrophobic packing with the antigen as well asbetter VL/VH pairing.

The epitope and paratope residues. FIG. 15 shows the 2D interaction mAbbetween IFN-ω and IFWM3421. The epitope residues are identical to thosein the M371 structure. The paratope residues are also almost identical(FIG. 15). However, as described above, the maturation process resultedin a number of structural and interaction differences, which likelyaccount for the improvement in binding affinity.

Example 17. Crystal Structure of IFWM3525 1 in Complex with IFN-ω T80E

Crystallization, X-ray data collection and structure determination wasdone essentially as described in Example 6.

The complex was prepared by mixing of IFN-ω with Fab of IFWM3525 inmolar ratio of 1.05:1.0 (excess IFN-ω, 1.92:1.12 mg), incubated at 4° C.overnight, and purified on Superdex 200 column equilibrated with 20 mmHEPES pH 7.5, 0.25 M NaCl, 10% glycerol, then concentrated to 9.79mg/ml. Crystals suitable for X-diffraction were obtained from 18% PEG3K, 0.2 M sodium citrate by MMS seeding with seeds from IFN-ω/Fab3186crystals.

For X-ray data collection, one crystal of IFN-ω/IFWM3525 complex wassoaked for a few seconds in a synthetic mother liquor (20% PEG 3350, 0.2M sodium citrate, 25% glycerol), and flash frozen in the liquidnitrogen. X-ray data were collected at APS (Argonne National Lab). Thediffraction data were processed with XDS¹⁰.

The structure of the IFN-ω/IFWM3525 complex was solved by molecularreplacement (MR) with Phaser. The search models for MR were the crystalstructure of IFN-ω/FabM371. The structure was then refined with PHENIXand model adjustments were carried out using COOT. All othercrystallographic calculations were performed with the CCP4 suite ofprograms. All molecular graphics were generated with PyMol. Thestructure refinement statistics are given in Table 31.

TABLE 31 IFNω/Fab IFWM3525 Crystal data Space group C2 Unit celldimensions a, b, c (Å) 169.53, 132.78, 144.19 α, β, γ (°) 90, 120.43, 90Asymmetric unit content 4 complex X-ray data Resolution (Å) 50-3.14(3.22-3.14)* Number of measured reflections 161,700 (9,097) Number ofunique reflections 47,615 (3,033) Completeness (%) 98.30 (85.1)R_(merge) 0.106 (0.877) <I/σ> 10.7 (1.6) B-factor (Wilson plot) (Å²)79.9 Refinement Resolution (Å) 47.9-3.14 (3.20-3.14) Number of reflsused in refinement 47,403 (2,685) Number of all atoms 14,880 Number ofsolvent molecules 0 Rcryst (%) 24.4 (37.1) Rfree (%) 28.4 (42.0) RMSDbond lengths (Å) 0.002 RMSD bond angles (°) 0.60 RMSD B-factormain-chain (Å²) 5.2 Mean B-factor (Å²) 88.9 Protein 88.9 Solvent N/AMolProbity [25] Clash score 3.2 Rotamer outliers (%) 0.4 Ramachandranfavored (%) 96.1 Ramachandran outliers (%) 0.3 Cβ deviation (>0.25 Å) 0*Values for high-resolution shell are in parentheses

The overall structure of the IFN-ω/IFWM35258 complex was very similar toIFN-ω/FabM371. The molecular models for the IFN-ω molecules includesresidues 23-39 and 119-153, corresponding to helical segment AB andhelices D and E. The helices A, B and C and the connecting loops aredisordered. These missing parts of the IFN-ω are likely due to limitedproteolysis as found for the M371 and M3421 complex structures. The Fabmolecular model contains residues from 1 to 213 for the light chain andfrom 1 to 222 for the heavy chain. The C-terminal 6xHis tag, inter-chaindisulfide bond and residues of 137-141 of the heavy chain aredisordered. No solvent water molecules were included due to lowdiffraction resolution.

FIG. 16 shows a 2-dimensional interaction map between IFN-ω and Fab ofIFWM3525. Epitope residues F27, L30, and R33 of the AB helix account forthe majority of the Ab/Ag interactions. Thus, this region of IFN-ωappears to constitute the main part of the epitope. Compared with theparental M371, the epitope contains two more residues from the helix Eof IFN-ω which form interactions with HCDR3 of IFWM3525.

IFWM3525 has broad binding specificity for IFNω and most of IFNαsubtypes. It does not bind IFNβ and IFNα-D/1. The sequence alignment ofIFNs (FIG. 9) indicates that IFWM3525 epitope residues are largelyconserved among the IFN-ω and IFN-α subtypes. In addition, structuralcomparison of the epitope residues in IFN-α (pdb code 2RH2, which wasre-built and refined using deposited data as only Cα trace was availablein PDB) and IFN-ω indicate the epitope residues have very similarbackbone and side chain structures. Thus, the sequence and structureconservations (or epitope conservation) likely are responsible for thebroad binding of IFNα/ω by IFWM3525.

Sequence Listing SEQ Amino acid sequence (or ID De-nucleotide sequence, as NO: Type Species scription applicable) 1 PRTHomo human IFNw CDLPQNHGLLSRNTLVLLHQMRRISPFLCLK sapiensDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQ QIFSLFHTERSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYF QGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGSS 2 PRT Homo hu IFNw  CDLPQNHGLLSRNTLVLLHQMRRISPFLCLKsapiens T80E  DRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMELLDQLHTGLHQQL QHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLS TNMQERLRSKDRDLGSS 3 PRT Chimp chimpCDLPQNHGLLSRNTLVLLHQMRRISPFLCLK IFNomega DRRDFRFPQEMVKGSQLQKAQVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQL QHLETCLLQVMGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLS TNMQERLRSKDRDLGSSRNDSH 4 PRT Cyno cynoCDLPQNHGLLSRNTLVLLHQMRRISPFLCLK IFNomega DRRDFRFPQEMVEGSQLQKAQVMSVLHEMLQQIFSLFHTEHSSAAWNTTLLDHLHTGLHRQL EHLETCLVQVMREGESAGAIRSPALTLRRYFQGIRVYLKEKKYSDCAWVVVRMEIMKSLFLS TNMQERLKSKDGDLGSS 5 PRT Homo alpha ACDLPQTHSLGSRRTLMLLAQMRKISLFSCLK sapiens DRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLN DLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLST NLQESLRSKE 6 PRT Homo IFN alphaCDLPQTHSLGNRRALILLAQMRRISPFSCLK sapiens B2DRHDFEFPQEEFDDKQFQKAQAISVLHEMIQ QTFNLFSTKDSSAALDETLLDEFYIELDQQLNDLESCVMQEVGVIESPLMYEDSILAVRKYF QRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE 7 PRT Homo alpha C CDLPQTHSLGNRRALILLGQMGRISPFSCLK sapiensDRHDFRIPQEEFDGNQFQKAQAISVLHEMIQ QTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYF QRITLYLIERKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD 8 PRT Homo alpha D CDLPETHSLDNRRTLMLLAQMSRISPSSCLM sapiensDRHDFGFPQEEFDGNQFQKAPAISVLHELIQ QIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNVDSILAVKKYF RRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE 9 PRT Homo alpha F CDLPQTHSLGNRRALILLAQMGRISPFSCLK sapiensDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQ QTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDMEACVIQEVGVEETPLMNVDSILAVKKYF QRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE 10 PRT Homo alpha G CDLPQTHSLSNRRTLMIMAQMGRISPFSCLK sapiensDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQ QTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEDTPLMNVDSILTVRKYF QRITLYLTEKKYSPCAWEVVRAEIMRSFSLSANLQERLRRKE 11 PRT Homo alpha H2 CNLSQTHSLNNRRTLMLMAQMRRISPFSCLK sapiensDRHDFEFPQEEFDGNQFQKAQAISVLHEMMQ QTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDLEACVIQEVGVEETPLMNEDSILAVKKYF QRITLYLMEKKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD 12 PRT Homo IFN-aI CDLPQTHSLGNRRALILLAQMGRISPFSCLK sapiensDRPDFGLPQEEFDGNQFQKTQAISVLHEMIQ QTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNNLEACVIQEVGMEETPLMNEDSILAVRKYF QRITLYLTEKKYSPCAWEVVRAEIMRSLSFSTNLQKILRRKD 13 PRT Homo alpha J1 CDLPQTHSLRNRRALILLAQMGRISPFSCLK sapiensDRHEFRFPEEEFDGHQFQKTQAISVLHEMIQ QTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDFILAVRKYF QRITLYLMEKKYSPCAWEVVRAEIMRSFSFSTNLKKGLRRKD 14 PRT Homo alpha K CDLPQTHSLGHRRTMMLLAQMRRISLFSCLK sapiensDRHDERFPQEEEDGNQFQKAEAISVLHEVIQ QTFNLFSTKDSSVAWDERLLDKLYTELYQQLNDLEACVMQEVWVGGTPLMNEDSILAVRKYF QRITLYLTEKKYSPCAWEVVRAEIMRSFSSSRNLQERLRRKE 15 PRT Homo alpha 4b CDLPQTHSLGNRRALILLAQMGRISHFSCLK sapiensDRHDFGFPEEEFDGHQFQKTQAISVLHEMIQ QTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVRKYF QRITLYLTEKKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD 16 PRT Homo alpha WA CDLPQTHSLGNRRALILLAQMGRISHFSCLK sapiensDRYDFGFPQEVFDGNQFQKAQAISAFHEMIQ QTFNLFSTKDSSAAWDETLLDKFYIELFQQLNDLEACVTQEVGVEEIALMNEDSILAVRKYF QRITLYLMGKKYSPCAWEVVRAEIMRSFSFSTNLQKGLRRKD 17 PRT Homo IFN-a2 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLK sapiensDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQ IFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQ RITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 18 PRT Homo IFN-a1 CDLPETHSLDNRRTLMLLAQMSRISPSSCLM sapiensDRHDFGFPQEEFDGNQFQKAPAISVLHELIQ QIFNLFTTKDSSAAWDEDLLDKFCTELYQQLDLEACVMQEERVGETPLMNADSILAVKKYFR NRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE 19 PRT Homo IFN-a4a CDLPQTHSLGNRRALILLAQMGRISHFSCLK sapiensDRHDFGFPEEEFDGHQFQKAQAISVLHEMIQ QTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYF QRITLYLTEKKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD 20 PRT Homo IFN-b MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYC sapiensLKDRMNFDIPEEIKQLQQFQKEDAALTIYEM LQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKR YYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN 21 PRT Artificial Signal MALTFYLLVALVVLSYKSFSSLG sequencepeptide 22 PRT Artificial Signal MARSFSLLMVVLVLSYKSICSLG sequencepeptide 23 PRT Artificial Signal MALPFALLMALVVLSCKSSCSLD sequencepeptide 24 PRT Artificial Signal MALSFSLLMAVLVLSYKSICSLG sequencepeptide 25 PRT Artificial Signal MALTFALLVALLVLSCKSSCSVG sequencepeptide 26 PRT Homo IFNAR1   1 mmvvllgatt lvlvavapwv lsaaaggknl kspqkvevdi iddnfilrwn rsdesvgnvtsapiens  61fsfdyqktgm dnwiklsgcq nitstkcnfs slklnvyeei klriraeken tsswyevdsf 121tpfrkaqigp pevhleaedk aivihispgt kdsvmwaldg lsftyslviw knssgveeri 181eniysrhkiy klspettycl kvkaalltsw kigvyspvhc ikttvenelp ppenievsvq 241nqnyvlkwdy tyanmtfqvq wlhaflkrnp gnhlykwkqi pdcenvkttq cvfpqnvfqk 301giyllrvgas dgnntsfwse eikfdteiqa fllppvfnir slsdsfhiyi gapkqsgntp 361viqdypliye iifwentsna erkiiekktd vtvpnlkplt vycvkaraht mdeklnkssv 421fsdavcektk pgntskiwli vgicialfal pfviyaakvf lrcinyvffp slkpssside 481yfseqplknl llstseeqie kcfiienist iatveetnqt dedhkkyssq tsqdsgnysn 541edesesktse elqqdfv 27 PRT Homo IFNAR2   1mllsqnafif rslnlvlmvy islvfgisyd spdytdesct fkislrnfrs ilswelknhssapiens  61ivpthytlly timskpedlk vvkncanttr sfcdltdewr stheayvtvl egfsgnttlf 121scshnfwlai dmsfeppefe ivgftnhinv mvkfpsivee elqfdlslvi eeqsegivkk 181hkpeikgnms gnftyiidkl ipntnycvsv ylehsdeqav iksplkctll ppgqesesae 241sakiggiitv flialvltst ivtlkwigyi clrnslpkvl rqglakgwna vaihrcshna 301lgsetpelkg ssclsfpssw dykraslcps d 28 PRT Artificial IFWH591EVQLVQSGAEVKKPGESLKISCKGSGYSFTS sequence YWIGWVRQMPGKGLEWMGIIDPSDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM YYCARHPGLNWAPDFDYWGQGTLVTVSS 29 PRTArtificial PH9L4 DIQMTQSPSSLSASVGDRVTITCRASQSISS sequenceYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK 30 PRT Artificial IFWH624 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSsequence YWIGWVRQMPGKGLEWMGIIDPSDSDTAYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHPGLNWAPDFDYWGQGTLVTVSS 31 PRT Artificial IFWH629EVQLVQSGAEVKKPGESLKISCKGSGYSFTS sequence YWIGWVRQMPGKGLEWMGIIDPSDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM YYCARHPGLAWAPDFDYWGQGTLVTVSS 32 PRTArtificial IFWL983 DIQMTQSPSSLSASVGDRVTITCRASQSIDG sequenceSLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYDFPLTFGQGTKVEIK 33 PRT Artificial IFWL991 DIQMTQSPSSLSASVGDRVTITCRASQSINRsequence FLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAIDLPFTFGQGTKVEIK 34 PRT Artificial IFWL992 DIQMTQSPSSLSASVGDRVTITCRASQSIGSsequence FLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPITFGQGTKVEIK 35 PRT Artificial IFWL997 DIQMTQSPSSLSASVGDRVTITCRASQSIGSsequence ALNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYDFPLTFGQGTKVEIK 36 PRT Artificial IFWL998 DIQMTQSPSSLSASVGDRVTITCRASQSISKsequence FLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSNTLPFTFGQGTKVEIK 37 PRT Artificial IFWL999 DIQMTQSPSSLSASVGDRVTITCRASQSIDEsequence FLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAHSFPLTFGQGTKVEIK 38 PRT Artificial IFWL1000DIQMTQSPSSLSASVGDRVTITCRASQSITN sequence FLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLD FPLTFGQGTKVEIK 39 PRT ArtificialIFWL1001 DIQMTQSPSSLSASVGDRVTITCRASQSIGD sequenceFLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQALDFPLTFGQGTKVEIK 40 PRT Artificial IFWL1004DIQMTQSPSSLSASVGDRVTITCRASQSIAE sequence FLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSID FPLTFGQGTKVEIK 41 PRT ArtificialIFWL1006 DIQMTQSPSSLSASVGDRVTITCRASQSIGG sequenceFLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPITFGQGTKVEIK 42 PRT Artificial IFWL1007DIQMTQSPSSLSASVGDRVTITCRASQSIGK sequence SLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD FPLTFGQGTKVEIK 43 PRT ArtificialIFWL1009 DIQMTQSPSSLSASVGDRVTITCRASQSIDD sequenceFLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSHTLPLTFGQGTKVEIK 44 PRT Artificial IFWL1010DIQMTQSPSSLSASVGDRVTITCRASQSIDG sequence ALNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFD FPLTFGQGTKVEIK 45 PRT ArtificialIFWL1013 DIQMTQSPSSLSASVGDRVTITCRASQSINN sequenceFLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSFNLPITFGQGTKVEIK 46 PRT Artificial IFWL1014DIQMTQSPSSLSASVGDRVTITCRASQSIDR sequence ALNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFD FPLTFGQGTKVEIK 47 PRT ArtificialIFWL1017 DIQMTQSPSSLSASVGDRVTITCRASQSITS sequenceSLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSFDLPLTFGQGTKVEIK 48 PRT Artificial IFWL1022DIQMTQSPSSLSASVGDRVTITCRASQSINE sequence FLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYS TPLTFGQGTKVEIK 49 PRT ArtificialIFWL1026 DIQMTQSPSSLSASVGDRVTITCRASQSISK sequenceFLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYDFPITFGQGTKVEIK 50 PRT Artificial IFWL1038DIQMTQSPSSLSASVGDRVTITCRASQSISE sequence YLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHS LPITFGQGTKVEIK 51 PRT ArtificialIFWL1041 DIQMTQSPSSLSASVGDRVTITCRASQSITG sequenceFLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSHDFPLTFGQGTKVEIK 52 PRT Artificial IFWL1047DIQMTQSPSSLSASVGDRVTITCRASQSING sequence VLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHD FPLTFGQGTKVEIK 53 PRT ArtificialIFWL1048 DIQMTQSPSSLSASVGDRVTITCRASQSIDG sequenceALNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAYDFPLTFGQGTKVEIK 54 PRT Artificial IFWL1051DIQMTQSPSSLSASVGDRVTITCRASQSIAD sequence FLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHS FPLTFGQGTKVEIK 55 PRT ArtificialIFWL1053 DIQMTQSPSSLSASVGDRVTITCRASQSITN sequenceHLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAHNFPLTFGQGTKVEIK 56 PRT Artificial IFWL1060DIQMTQSPSSLSASVGDRVTITCRASQSIRN sequence SLNWYQQKPGKAPKLLIKWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLYD WPLTFGQGTKVEIK 57 PRT ArtificialIFWL1063 DIQMTQSPSSLSASVGDRVTITCRASQSIAN sequenceNNLNWYQQKPGKAPKLLIHWASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQGYDTPFTFGQGTKVEIK 58 PRT Artificial IFWL1064DIQMTQSPSSLSASVGDRVTITCRASQSINN sequence LNWYQQKPGKAPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYDT PFTFGQGTKVEIK 59 PRT Artificial IFWL1067DIQMTQSPSSLSASVGDRVTITCRASQSIRN sequence NNLNWYQQKPGKAPKLLIHWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DTPFTFGQGTKVEIK 60 PRT ArtificialIFWL1071 DIQMTQSPSSLSASVGDRVTITCRASQSIRN sequenceNSLNWYQQKPGKAPKLLIYGASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQDYNWPITFGQGTKVEIK 61 PRT Artificial IFWL1073DIQMTQSPSSLSASVGDRVTITCRASQSIDN sequence SYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGW DWPLTFGQGTKVEIK 62 PRT ArtificialIFWL1074 DIQMTQSPSSLSASVGDRVTITCRASQSIAN sequenceTNLNWYQQKPGKAPKLLIHWASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWYDNPLTFGQGTKVEIK 63 PRT Artificial IFWL1076DIQMTQSPSSLSASVGDRVTITCRASQSIDN sequence NNLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DWPLTFGQGTKVEIK 64 PRT ArtificialIFWL1082 DIQMTQSPSSLSASVGDRVTITCRASQSIRN sequenceNSLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQDYNWPLTFGQGTKVEIK 65 PRT Artificial IFWL1084DIQMTQSPSSLSASVGDRVTITCRASQSINY sequence LNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHDW PITFGQGTKVEIK 66 PRT Artificial IFWL1085DIQMTQSPSSLSASVGDRVTITCRASQSIRN sequence NYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DTPLTFGQGTKVEIK 67 PRT ArtificialIFWL1087 DIQMTQSPSSLSASVGDRVTITCRASQSISN sequenceSNLNWYQQKPGKAPKLLIHWASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWYDHPLTFGQGTKVEIK 68 PRT Artificial IFWL1091DIQMTQSPSSLSASVGDRVTITCRASQSIRN sequence TNLNWYQQKPGKAPKLLIHWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DTPFTFGQGTKVEIK 69 PRT ArtificialIFWL1093 DIQMTQSPSSLSASVGDRVTITCRASQSIAN sequenceNDLNWYQQKPGKAPKLLIHWASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQDYDWPLTFGQGTKVEIK 70 PRT Artificial IFWL1049DIQMTQSPSSLSASVGDRVTITCRASQSIAG sequence FLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYS IPITFGQGTKVEIK 71 PRT Artificial IFWL984DIQMTQSPSSLSASVGDRVTITCRASQSIDG sequence FLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 72 DNA Artificial cDNA ofGATATTCAGATGACCCAGAGCCCGAGCAGCC sequence IFWL984TGAGCGCGAGCGTGGGCGATCGCGTGACCAT TACCTGCCGCGCGAGCCAGAGCATTGATGGGTTCCTGAACTGGTATCAGCAGAAACCGGGCA AAGCGCCGAAACTGCTGATTTATTTCGCGAGCAGCCTGCAGAGCGGCGTGCCGAGCCGCTTT AGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTT TGCGACCTATTATTGCCAGCAGTCCTACGACCTCCCGATTACATTTGGCCAGGGCACCAAAG TGGAAATTAAA 73 PRT Artificial IFWL1136DIQMTQSPSSLSASVGDRVTITCRASQSIEG sequence ALNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD FPLTFGQGTKVEIK 74 PRT ArtificialIFWL1144 DIQMTQSPSSLSASVGDRVTITCRASQSIEG sequenceYLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAYDFPLTFGQGTKVEIK 75 PRT Artificial IFWL1148DIQMTQSPSSLSASVGDRVTITCRASQSISS sequence ALNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD FPLTFGQGTKVEIK 76 PRT Artificial LCDR1QSIADF sequence 77 PRT Artificial LCDR1 QSIAEF sequence 78 PRTArtificial LCDR1 QSIANNN sequence 79 PRT Artificial LCDR1 QSIANTNsequence 80 PRT Artificial LCDR1 QSIDGA sequence 81 PRT Artificial LCDR1QSIDGF sequence 82 PRT Artificial LCDR1 QSIDNSY sequence 83 PRTArtificial LCDR1 QSIDRA sequence 84 PRT Artificial LCDR1 QSIEGA sequence85 PRT Artificial LCDR1 QSIGDF sequence 86 PRT Artificial LCDR1 QSIGKSsequence 87 PRT Artificial LCDR1 QSIGSA sequence 88 PRT Artificial LCDR1QSINGV sequence 89 PRT Artificial LCDR1 QSIRNTN sequence 90 PRTArtificial LCDR1 QSISSA sequence 91 PRT Artificial LCDR1 QSISSF sequence92 DNA Artificial cDNA of GACATCCAAATGACGCAGTCTCCGAGCTCTC sequenceIFWL1164 TGAGCGCATCCGTGGGCGATCGCGTAACTAT CACTTGTCGCGCCTCCCAGAGCATTGATAACTCCTATCTCAATTGGTATCAACAAAAACCGG GTAAGGCACCGAAACTGCTGATTTACGGAGCGTCCTCTCTGCAGTCCGGTGTGCCGTCCCGT TTCTCCGGCAGCGGTTCTGGTACCGATTTCACGCTGACCATCAGCTCTCTGCAACCGGAGGA CTTTGCTACGTACTACTGCCAACAGGGCTACGATTTCCCTCTCACATTCGGCCAAGGTACCA AAGTGGAAATTAAA 93 PRT Artificial LCDR2FAS sequence 94 PRT Artificial LCDR2 GAS sequence 95 PRT ArtificialLCDR2 WAS sequence 96 PRT Artificial LCDR3 QQALDFPLT sequence 97 PRTArtificial LCDR3 QQAYDFPLT sequence 98 PRT Artificial LCDR3 QQGWDWPLTsequence 99 PRT Artificial LCDR3 QQGYDFPLT sequence 100 PRT ArtificialLCDR3 QQGYDTPFT sequence 101 PRT Artificial LCDR3 QQSFDFPLT sequence 102PRT Artificial LCDR3 QQSHDFPLT sequence 103 PRT Artificial LCDR3QQSHSFPLT sequence 104 PRT Artificial LCDR3 QQSIDFPLT sequence 105 PRTArtificial LCDR3 QQSYDFPLT sequence 106 PRT Artificial LCDR3 QQSYDLPITsequence 107 PRT Artificial LCDR3 QQWYDNPLT sequence 108 DNA ArtificialcDNA of GATATTCAGATGACCCAGAGCCCGAGCAGCC sequence IFWL1048TGAGCGCGAGCGTGGGCGATCGCGTGACCAT TACCTGCCGCGCGAGCCAGAGCATCGATGGCGCCCTGAACTGGTATCAGCAGAAACCGGGCA AAGCGCCGAAACTGCTGATTTATTTCGCGAGCAGCCTGCAGAGCGGCGTGCCGAGCCGCTTT AGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTT TGCGACCTATTATTGCCAGCAGGCCTACGACTTTCCGTTGACATTTGGCCAGGGCACCAAAG TGGAAATTAAA 109 PRT Artificial HCDR1GYSFTSYW 110 DNA Artificial cDNA of GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGsequence IFWH591 TGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGCTTCACCAGC TACTGGATCGGCTGGGTGCGGCAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCGA CCCCAGCGACAGCGACACCCGGTACAGCCCCAGCTTCCAGGGCCAGGTGACCATCAGCGCCG ACAAGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCCATG TACTACTGCGCCCGGCACCCCGGCCTGAACTGGGCCCCCGACTTCGACTACTGGGGCCAGGG CACCCTGGTGACCGTGAGCAGC 111 DNAArtificial HCDR2 IAPSDSDT sequence 112 DNA Artificial HCDR2 IDASDSDTsequence 113 DNA Artificial HCDR2 IDPSDSDT sequence 114 PRT ArtificialHCDR2 IX₁₁X₁₂SDSDT; whrein consensus X₁₁ is D or A; and sequenceX₁₂ is P or A. mAbs neutralize at least 3 IFNalphas 115 PRT ArtificialHCDR3 ARHPGLAWAPDFDY 116 PRT Artificial HCDR3 ARHPGLNWAPDFDY 117 DNAArtificial cDNA of GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGG sequence IFWH617TGAAGAAGCCCGGCGAGAGCCTGAAGATCAG CTGCAAGGGCAGCGGCTACAGCTTCACCAGCTACTGGATCGGCTGGGTGCGGCAGATGCCCG GCAAGGGCCTGGAGTGGATGGGCATCATCGACGCCAGCGACAGCGACACCCGGTACAGCCCC AGCTTCCAGGGCCAGGTGACCATCAGCGCCGACAAGAGCATCAGCACCGCCTACCTGCAGTG GAGCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGCGCCCGGCACCCCGGCCTGAACT GGGCCCCCGACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC 118 PRT Artificial LCDR1 QSIX₁X₂X₃X₄; whereinconsensus X₁ is G, D, A, R, E, S, or N; sequenceX₂ is D, G, N, S, R, E or K; mAbs X₃ is F, A, N, T, S or V; neutralizeX₄ is Y, N or deleted. at least 3 IFNalphas 119 PRT Artificial LCDR2X₅AS; wherein consensus X₅ is F, W or G. sequence mAbs neutralizeat least 3 IFNalphas 120 PRT Artificial LCDR3 QQX₆X₇X₈X₉PX₁₀T; whereinconsensus X₆ is A, G, S or W; sequence X₇ is L, Y, H, W, F or I; mAbsX₈ is D or S; neutralize X₉ is F, T, L, N or W; and at least 3X₁₀ is L, F or I. IFNalphas 121 PRT Artificial HCDR3ARHPGLX₁₃WAPDFDY; wherein consensus X₁₃ is A or N. sequence mAbsneutralize at least 3 IFNalphas 122 DNA Artificial cDNA ofGAGGTGCAGCTGGTGCAGAGCGGCGCCGAGG IFWH629 TGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGCTTCACCAGC TACTGGATCGGCTGGGTGCGGCAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCGA CCCCAGCGACAGCGACACCCGGTACAGCCCCAGCTTCCAGGGCCAGGTGACCATCAGCGCCG ACAAGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCCATG TACTACTGCGCCCGGCACCCCGGCCTGGCCTGGGCCCCCGACTTCGACTACTGGGGCCAGGG CACCCTGGTGACCGTGAGCAGC 123 PRTArtificial IFWL1112 DIQMTQSPSSLSASVGDRVTITCRASQSISGFLNWYQQKPGKAPKLLIYFASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYDLPITFGQGTKVEIK 124 PRT Artificial IFWL1113DIQMTQSPSSLSASVGDRVTITCRASQSIEG FLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 125 PRT ArtificialIFWL1114 DIQMTQSPSSLSASVGDRVTITCRASQSIDG YLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 126 PRT ArtificialIFWL1115 DIQMTQSPSSLSASVGDRVTITCRASQSIDG FLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 127 PRT ArtificialIFWL1117 DIQMTQSPSSLSASVGDRVTITCRASQSIDG FLNWYQQKPGKAPKLLIYIASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 128 PRT ArtificialIFWL1118 DIQMTQSPSSLSASVGDRVTITCRASQSIDG FLNWYQQKPGKAPKLLIYLASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 129 PRT ArtificialIFWL1119 DIQMTQSPSSLSASVGDRVTITCRASQSIDG FLNWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 130 PRT ArtificialIFWL1120 DIQMTQSPSSLSASVGDRVTITCRASQSIEG YLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 131 PRT ArtificialIFWL1121 DIQMTQSPSSLSASVGDRVTITCRASQSIEG FLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 132 PRT ArtificialIFWL1122 DIQMTQSPSSLSASVGDRVTITCRASQSIEG FLNWYQQKPGKAPKLLIYIASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 133 PRT ArtificialIFWL1123 DIQMTQSPSSLSASVGDRVTITCRASQSIEG FLNWYQQKPGKAPKLLIYLASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 134 PRT ArtificialIFWL1124 DIQMTQSPSSLSASVGDRVTITCRASQSIEG FLNWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 135 PRT ArtificialIFWL1125 DIQMTQSPSSLSASVGDRVTITCRASQSISS FLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 136 PRT ArtificialIFWL1126 DIQMTQSPSSLSASVGDRVTITCRASQSISS YLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 137 PRT ArtificialIFWL1129 DIQMTQSPSSLSASVGDRVTITCRASQSIGD FLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 138 PRT ArtificialIFWL1173 DIQMTQSPSSLSASVGDRVTITCRASQSIEG YLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 139 PRT ArtificialIFWL1174 DIQMTQSPSSLSASVGDRVTITCRASQSIEG FLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 140 PRT ArtificialIFWL1175 DIQMTQSPSSLSASVGDRVTITCRASQSISS FLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYD LPITFGQGTKVEIK 141 PRT ArtificialIFWL1135 DIQMTQSPSSLSASVGDRVTITCRASQSISG ALNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD FPLTFGQGTKVEIK 142 PRT ArtificialIFWL1137 DIQMTQSPSSLSASVGDRVTITCRASQSIDG YLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD FPLTFGQGTKVEIK 143 PRT ArtificialIFWL1143 DIQMTQSPSSLSASVGDRVTITCRASQSIDG ALNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD LPLTFGQGTKVEIK 144 PRT ArtificialIFWL1149 DIQMTQSPSSLSASVGDRVTITCRASQSISS YLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD FPLTFGQGTKVEIK 145 PRT ArtificialIFWL1152 DIQMTQSPSSLSASVGDRVTITCRASQSISS ALNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD LPLTFGQGTKVEIK 146 PRT ArtificialIFWL1155 DIQMTQSPSSLSASVGDRVTITCRASQSISS YLNWYQQKPGKAPKLLIYFASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD LPLTFGQGTKVEIK 147 PRT ArtificialIFWL1161 DIQMTQSPSSLSASVGDRVTITCRASQSIGD FLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYD LPLTFGQGTKVEIK 148 PRT ArtificialIFWL1162 DIQMTQSPSSLSASVGDRVTITCRASQSIDN SYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DWPLTFGQGTKVEIK 149 PRT ArtificialIFWL1163 DIQMTQSPSSLSASVGDRVTITCRASQSIDN SYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGW DFPLTFGQGTKVEIK 150 PRT ArtificialIFWL1164 DIQMTQSPSSLSASVGDRVTITCRASQSIDN SYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DFPLTFGQGTKVEIK 151 PRT ArtificialIFWL1176 DIQMTQSPSSLSASVGDRVTITCRASQSIDQ SYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DFPLTFGQGTKVEIK 152 PRT ArtificialIFWL1177 DIQMTQSPSSLSASVGDRVTITCRASQSIDT SYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DFPLTFGQGTKVEIK 153 PRT ArtificialIFWL1178 DIQMTQSPSSLSASVGDRVTITCRASQSIDN TYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGY DFPLTFGQGTKVEIK 154 PRT Artificial LCDR3QQSYDFPL 155 PRT Homo IGHV5-51 EVQLVQSGAEVKKPGESLKISCKGSGYSFTS sapiensYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAV YYCAR156 PRT Homo IGKV1D-39 DIQMTQSPSSLSASVGDRVTITCRASQSISS sapiensYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK 157 PRT Artificial IFWH615EVQLVQSGAEVKKPGESLKISCKGSGYSFTS sequence YWIGWVRQMPGKGLEWMGIIAPSDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM YYCARHPGLNWAPDFDYWGQGTLVTVSS 158 PRTArtificial IFWH617 EVQLVQSGAEVKKPGESLKISCKGSGYSFTS sequenceYWIGWVRQMPGKGLEWMGIIDASDSDTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHPGLNWAPDFDYWGQGTLVTVSS 159 PRT Artificial LCDR1QSIX₁₄X₁₅X₁₆X₁₇; wherein consensus X₁₄ is G, D, A, E, S, or N; sequenceX₁₅ is D, G, N, S or R; mAbs X₁₆ is F, A, N, S or V; and neutralizeX₁₇ is Y, N or deleted. at least 6 IFNalphas 160 PRT Artificial LCDR3QQX₁₈X₁₉X₂₀X₂₁PX₂₂T; wherein consensus X₁₈ is A, G or S; sequenceX₁₉ is Y, H, W or F; mAbs X₂₀ is D or S; neutralizeX₂₁ is F, T, L or W; and at least 6 X₂₂ is L, F or I. IFNalphas 161 PRTArtificial LCDR1 QSIX₂₃X₂₄X₂₅X₂₆; wherein consensus X₂₃ is A or D;sequence X₂₄ is N or G; mAbs X₂₅ is F, N or S; and neutralizeX₂₆ is Y, N or deleted. at least 10 IFNalphas 162 PRT Artificial LCDR3QQX₂₇X₂₈X₂₉X₃₀PX₃₁T; wherein consensus X₂₇ is G or S; sequence X₂₈ is Y;mAbs X₂₉ is D; neutralize X₃₀ is F, T or L; and at least 10X₃₁ is L, F or I. IFNalphas

We claim: 1) An isolated polynucleotide encoding an antibody comprisinga heavy chain variable region and a light chain variable region, theheavy chain variable region comprising heavy chain complementaritydetermining region (HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) amino acidsequences of SEQ ID NOs: 109, 113 and 116, respectively, and the lightchain variable region comprising light chain complementarity determiningregion (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) amino acid sequences ofSEQ ID NOs: 82, 94 and 99, respectively. 2) A vector comprising thepolynucleotide of claim
 1. 3) A host cell comprising the vector of claim2. 4) A method of producing an antibody, comprising culturing the hostcell of claim 3 in conditions that the antibody is expressed, andrecovering the antibody produced by the host cell. 5) An isolatedpolynucleotide encoding an antibody comprising a heavy chain variableregion (VH) amino acid sequence of SEQ ID NO: 28 and a light chainvariable region (VL) amino acid sequences of SEQ ID NO:
 150. 6) A vectorcomprising the polynucleotide of claim
 5. 7) A host cell comprising thevector of claim
 6. 8) A method of producing an antibody, comprisingculturing the host cell of claim 7 in conditions that the antibody isexpressed, and recovering the antibody produced by the host cell.